Well-liked 405 secondary antibodies

[Joe Lebowitz]

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Hello all,

We have had some reproducibly disappointing results with Alexa Fluor 405
tagged secondary antibodies.

Does anyone have a go-to secondary in this color other than the AF? We are
trying to multiplex as many channels as possible and this is the only one we
struggle to get good brightness/SNR with.

Thanks!

joe

--------------------------------  

Joe Lebowitz
Ph.D. Candidate
NINDS Pre-doctoral Fellow
Khoshbouei Lab
UF Department of Neuroscience
[phone]  

 

[kspencer007]

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Hi Joe;
        I've had good success with Becton Dickinson BV dyes. BV421 is the best blue fluorescent dye I've tried.
        http://www.bdbiosciences.com/us/solrSearch?text=bv421&x=0&y=0

Kathy

The Scripps Research Institute
Dept of Molecular and Cellular Neuroscience
10550 N. Torrey Pines Road
DNC 216
La Jolla, Ca 92037





-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Joe Lebowitz
Sent: Thursday, May 02, 2019 1:17 PM
To: [hidden email]
Subject: Well-liked 405 secondary antibodies

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Hello all,

We have had some reproducibly disappointing results with Alexa Fluor 405 tagged secondary antibodies.

Does anyone have a go-to secondary in this color other than the AF? We are trying to multiplex as many channels as possible and this is the only one we struggle to get good brightness/SNR with.

Thanks!

joe

--------------------------------  

Joe Lebowitz
Ph.D. Candidate
NINDS Pre-doctoral Fellow
Khoshbouei Lab
UF Department of Neuroscience
[phone]  

 

[Armstrong, Brian]

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Hi Joe, we struggle with this also. I imagine it is a common complaint. Unfortunately, I would have to say that, imaging blue fluorophores with a 405nm LASER on a Confocal, we have had the best luck with Alexa 405.
Sorry,

Brian Armstrong PhD
Associate Research Professor
Developmental and Stem Cell Biology
Diabetes and Metabolic Diseases
Director, Light Microscopy Core
Beckman Research Institute, City of Hope



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joe Lebowitz
Sent: Thursday, May 02, 2019 1:17 PM
To: [hidden email]
Subject: Well-liked 405 secondary antibodies

[Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.]





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*****

Hello all,

We have had some reproducibly disappointing results with Alexa Fluor 405 tagged secondary antibodies.

Does anyone have a go-to secondary in this color other than the AF? We are trying to multiplex as many channels as possible and this is the only one we struggle to get good brightness/SNR with.

Thanks!

joe

--------------------------------

Joe Lebowitz
Ph.D. Candidate
NINDS Pre-doctoral Fellow
Khoshbouei Lab
UF Department of Neuroscience
[phone]



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[Craig Brideau]

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What ever happened to Brilliant Violet? I remember a bit of marketing about
it a while ago, but never got around to trying it myself. Did anyone get a
chance to give it a go?

Craig

On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello all,
>
> We have had some reproducibly disappointing results with Alexa Fluor 405
> tagged secondary antibodies.
>
> Does anyone have a go-to secondary in this color other than the AF? We are
> trying to multiplex as many channels as possible and this is the only one
> we
> struggle to get good brightness/SNR with.
>
> Thanks!
>
> joe
>
> --------------------------------
>
> Joe Lebowitz
> Ph.D. Candidate
> NINDS Pre-doctoral Fellow
> Khoshbouei Lab
> UF Department of Neuroscience
> [phone]
>
>
>

[Jacqueline Ross]

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Hi Joe,

We also find that AlexaFluor 405 performs the best for our confocal systems as all except one have 405nm lasers. One other comment I would make is that depending on which filter set you have available for observation, the signal may look very weak to your eye (as it's violet) but may be much better than you think. I have often been pleasantly surprised by the signal strength of staining with this fluorophore when I switch over to confocal. I would also suggest increasing the concentration of the secondary and putting your best primary antibody into this channel if possible.

Cheers,

Jacqui
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Joe Lebowitz [[hidden email]]
Sent: 03 May 2019 08:16
To: [hidden email]
Subject: Well-liked 405 secondary antibodies

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*****

Hello all,

We have had some reproducibly disappointing results with Alexa Fluor 405
tagged secondary antibodies.

Does anyone have a go-to secondary in this color other than the AF? We are
trying to multiplex as many channels as possible and this is the only one we
struggle to get good brightness/SNR with.

Thanks!

joe

--------------------------------

Joe Lebowitz
Ph.D. Candidate
NINDS Pre-doctoral Fellow
Khoshbouei Lab
UF Department of Neuroscience
[phone]

[Alison J. North]

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Hi Joe,

I really like DyLight 405 both for confocal and 3D-SIM.  It's very picky
regarding anti-fades though - use Prolong Diamond (uncured) and it is
just fine (not so good with Prolong Gold). Just to warn you, if you look
down the microscope visually you will think the staining hasn't worked,
the autofluorescence overwhelms the dye, but when you start imaging it's
totally different.

Good luck!

Alison


On 5/2/2019 4:16 PM, Joe Lebowitz wrote:

> *****
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> *****
>
> Hello all,
>
> We have had some reproducibly disappointing results with Alexa Fluor 405
> tagged secondary antibodies.
>
> Does anyone have a go-to secondary in this color other than the AF? We are
> trying to multiplex as many channels as possible and this is the only one we
> struggle to get good brightness/SNR with.
>
> Thanks!
>
> joe
>
> --------------------------------
>
> Joe Lebowitz
> Ph.D. Candidate
> NINDS Pre-doctoral Fellow
> Khoshbouei Lab
> UF Department of Neuroscience
> [phone]
>
>  

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office    ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489

[Jeffrey Carmichael]

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Craig,

You've probably heard some of this from me, and I know that George McNamara
has also been impressed with and written of the Brilliant Horizon fluors
from BD (BD owns this from Sirigen purchase).

Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:

"Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
Hybridization."
https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization

BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
25-fold brighter than CFP (depending on who is measuring...)

The problem remains the availability of reagents, as BD has decided that
their business model is better served by reserving these reagents for their
custom-conjugated Ab's for flow.....But I believe that BD still entertains
custom requests for their reagents.

IMHO, directly conjugated, primary Ab's are the best way to leverage these
fluors because they're so bright that they don't need amplification. This
removes the problem of species specificity....Why wouldn't one try this???

Here's a link to a presentation that illustrates the efficacy of this
approach of filling the gap of violet & blue fluors.  Given the headlong
stampede towards increased multiplexing, I don't understand how these
fluors could be overlooked:
https://www.chroma.com/5-channel-fluorescence-imaging-simplified

I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
Technology has no formal relationship with BD Biosciences and receives no
credit for purchases of related products. Chroma Technology profits only
from sales of filter sets which are used to image these fluors.

Jeff

*Jeff Carmichael*

*Director of Marketing*

*[hidden email] <[hidden email]> | 802-428-2528*



On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> What ever happened to Brilliant Violet? I remember a bit of marketing about
> it a while ago, but never got around to trying it myself. Did anyone get a
> chance to give it a go?
>
> Craig
>
> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hello all,
> >
> > We have had some reproducibly disappointing results with Alexa Fluor 405
> > tagged secondary antibodies.
> >
> > Does anyone have a go-to secondary in this color other than the AF? We
> are
> > trying to multiplex as many channels as possible and this is the only one
> > we
> > struggle to get good brightness/SNR with.
> >
> > Thanks!
> >
> > joe
> >
> > --------------------------------
> >
> > Joe Lebowitz
> > Ph.D. Candidate
> > NINDS Pre-doctoral Fellow
> > Khoshbouei Lab
> > UF Department of Neuroscience
> > [phone]
> >
> >
> >
>

--
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*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>                             

[James Muller]

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BV421 is good but I always use it for highly expressed antigens. Also since it is a polymer dye it does not penetrate thick tissue sections well so some optimization needs to be done.

All the best,

JM

> On May 2, 2019, at 4:45 PM, Armstrong, Brian <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Joe, we struggle with this also. I imagine it is a common complaint. Unfortunately, I would have to say that, imaging blue fluorophores with a 405nm LASER on a Confocal, we have had the best luck with Alexa 405.
> Sorry,
>
> Brian Armstrong PhD
> Associate Research Professor
> Developmental and Stem Cell Biology
> Diabetes and Metabolic Diseases
> Director, Light Microscopy Core
> Beckman Research Institute, City of Hope
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joe Lebowitz
> Sent: Thursday, May 02, 2019 1:17 PM
> To: [hidden email]
> Subject: Well-liked 405 secondary antibodies
>
> [Attention: This email came from an external source. Do not open attachments or click on links from unknown senders or unexpected emails.]
>
>
>
>
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello all,
>
> We have had some reproducibly disappointing results with Alexa Fluor 405 tagged secondary antibodies.
>
> Does anyone have a go-to secondary in this color other than the AF? We are trying to multiplex as many channels as possible and this is the only one we struggle to get good brightness/SNR with.
>
> Thanks!
>
> joe
>
> --------------------------------
>
> Joe Lebowitz
> Ph.D. Candidate
> NINDS Pre-doctoral Fellow
> Khoshbouei Lab
> UF Department of Neuroscience
> [phone]
>
>
>
> ------------------------------------------------------------
> -SECURITY/CONFIDENTIALITY WARNING-
>
> This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (LCP301)
> ------------------------------------------------------------

[George McNamara]

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Hi Craig, Joe et al,

as previously noted in another reply, BV421 = Brilliant Violet 421,
works fine on 405 nm laser. Available from BD Biosciences (acquired the
manufacturer, Sirigen, for the Brilliants), and BioLegend.

We have a use with a CD## direct labeled primary antibody added to mouse
leukocytes, imaged live on our Leica SP8 confocal microscope (HyD1, 2nd
gen hybrid detector), including both time series (100 min) and Z-series.

I note BD Biosciences now has BUVs (excite well ~350 nm), a few BB's
(excite 488 nm) and one BYG (excite ~561 nm).

I have suggested to a number of users that they move on from DAPI and
Hoechst to BioLegend's Zombie NIR, excite ok ~640 nm, emission peak ~750
nm ... BV421-antibody is narrow emission spectrum, bright, and has lots
of antibodies ... DAPI and Hoechst a broad spectrum, one trick fluors.

enjoy,

George


On 5/2/2019 4:47 PM, Craig Brideau wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> What ever happened to Brilliant Violet? I remember a bit of marketing about
> it a while ago, but never got around to trying it myself. Did anyone get a
> chance to give it a go?
>
> Craig
>
> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hello all,
>>
>> We have had some reproducibly disappointing results with Alexa Fluor 405
>> tagged secondary antibodies.
>>
>> Does anyone have a go-to secondary in this color other than the AF? We are
>> trying to multiplex as many channels as possible and this is the only one
>> we
>> struggle to get good brightness/SNR with.
>>
>> Thanks!
>>
>> joe
>>
>> --------------------------------
>>
>> Joe Lebowitz
>> Ph.D. Candidate
>> NINDS Pre-doctoral Fellow
>> Khoshbouei Lab
>> UF Department of Neuroscience
>> [phone]
>>
>>
>>

[George McNamara]

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Hi Jeff,

Excellent post.

BD business model limitation ... can be dealt with. And the fluorescence
moicroscopy community would be well served by using as many "flow
cytometry" compatible reagents as possible.

BD Biosciences and BioLegend each sell BV421 streptavidin conjugates

http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/second-step-reagents/avidinstreptavidin/bv421-streptavidin/p/563259

https://www.biolegend.com/en-us/products/brilliant-violet-421-streptavidin-7297

I note that streptavidin has four binding sites for biotin, so may lose
some efficiency in conjugation = need to optimize amount of BV-SA :
biotin-mAb ... ideally get 1:1 conjugation, but if a complex forms.

Maybe best counter to BD's business model is to use Fab (or scFv)
instead of full length IgG. Or VHH nanobodies.

enjoy,

George


On 5/2/2019 7:02 PM, Jeff Carmichael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Craig,
>
> You've probably heard some of this from me, and I know that George McNamara
> has also been impressed with and written of the Brilliant Horizon fluors
> from BD (BD owns this from Sirigen purchase).
>
> Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
>
> "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> Hybridization."
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
>
> BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> 25-fold brighter than CFP (depending on who is measuring...)
>
> The problem remains the availability of reagents, as BD has decided that
> their business model is better served by reserving these reagents for their
> custom-conjugated Ab's for flow.....But I believe that BD still entertains
> custom requests for their reagents.
>
> IMHO, directly conjugated, primary Ab's are the best way to leverage these
> fluors because they're so bright that they don't need amplification. This
> removes the problem of species specificity....Why wouldn't one try this???
>
> Here's a link to a presentation that illustrates the efficacy of this
> approach of filling the gap of violet & blue fluors.  Given the headlong
> stampede towards increased multiplexing, I don't understand how these
> fluors could be overlooked:
> https://www.chroma.com/5-channel-fluorescence-imaging-simplified
>
> I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> Technology has no formal relationship with BD Biosciences and receives no
> credit for purchases of related products. Chroma Technology profits only
> from sales of filter sets which are used to image these fluors.
>
> Jeff
>
> *Jeff Carmichael*
>
> *Director of Marketing*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
>
>
> On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> What ever happened to Brilliant Violet? I remember a bit of marketing about
>> it a while ago, but never got around to trying it myself. Did anyone get a
>> chance to give it a go?
>>
>> Craig
>>
>> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hello all,
>>>
>>> We have had some reproducibly disappointing results with Alexa Fluor 405
>>> tagged secondary antibodies.
>>>
>>> Does anyone have a go-to secondary in this color other than the AF? We
>> are
>>> trying to multiplex as many channels as possible and this is the only one
>>> we
>>> struggle to get good brightness/SNR with.
>>>
>>> Thanks!
>>>
>>> joe
>>>
>>> --------------------------------
>>>
>>> Joe Lebowitz
>>> Ph.D. Candidate
>>> NINDS Pre-doctoral Fellow
>>> Khoshbouei Lab
>>> UF Department of Neuroscience
>>> [phone]
>>>
>>>
>>>

[0000001ed7f52e4a-dmarc-request]

*****
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*****

Interesting topic.

BD Biosciences and BioLegend each sell BV421 streptavidin conjugates 


Does this work? From my days in chemistry I remember that streptavidin sticks to almost anything, not what you want to combine with your highly specific antibody. Isn't t this why they developed neutravidin?

Maybe best counter to BD's business model is to use Fab (or scFv)
 instead of full length IgG. Or VHH nanobodies.


We used single Fab a lot for single molecule tracking and STORM. But they come off very easily, while you have less problems with inaccessible binding sites, labelling can be quite inefficient.
Best wishes
Andreas


-----Original Message-----
From: George McNamara <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Fri, 3 May 2019 5:08
Subject: Re: Well-liked 405 secondary antibodies **COMMERCIAL RESPONSE**

*****
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*****

Hi Jeff,

Excellent post.

BD business model limitation ... can be dealt with. And the fluorescence
moicroscopy community would be well served by using as many "flow
cytometry" compatible reagents as possible.

BD Biosciences and BioLegend each sell BV421 streptavidin conjugates

http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/second-step-reagents/avidinstreptavidin/bv421-streptavidin/p/563259

https://www.biolegend.com/en-us/products/brilliant-violet-421-streptavidin-7297

I note that streptavidin has four binding sites for biotin, so may lose
some efficiency in conjugation = need to optimize amount of BV-SA :
biotin-mAb ... ideally get 1:1 conjugation, but if a complex forms.

Maybe best counter to BD's business model is to use Fab (or scFv)
instead of full length IgG. Or VHH nanobodies.

enjoy,

George


On 5/2/2019 7:02 PM, Jeff Carmichael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Craig,
>
> You've probably heard some of this from me, and I know that George McNamara
> has also been impressed with and written of the Brilliant Horizon fluors
> from BD (BD owns this from Sirigen purchase).
>
> Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
>
> "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> Hybridization."
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
>
> BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> 25-fold brighter than CFP (depending on who is measuring...)
>
> The problem remains the availability of reagents, as BD has decided that
> their business model is better served by reserving these reagents for their
> custom-conjugated Ab's for flow.....But I believe that BD still entertains
> custom requests for their reagents.
>
> IMHO, directly conjugated, primary Ab's are the best way to leverage these
> fluors because they're so bright that they don't need amplification. This
> removes the problem of species specificity....Why wouldn't one try this???
>
> Here's a link to a presentation that illustrates the efficacy of this
> approach of filling the gap of violet & blue fluors.  Given the headlong
> stampede towards increased multiplexing, I don't understand how these
> fluors could be overlooked:
> https://www.chroma.com/5-channel-fluorescence-imaging-simplified
>
> I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> Technology has no formal relationship with BD Biosciences and receives no
> credit for purchases of related products. Chroma Technology profits only
> from sales of filter sets which are used to image these fluors.
>
> Jeff
>
> *Jeff Carmichael*
>
> *Director of Marketing*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
>
>
> On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> What ever happened to Brilliant Violet? I remember a bit of marketing about
>> it a while ago, but never got around to trying it myself. Did anyone get a
>> chance to give it a go?
>>
>> Craig
>>
>> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hello all,
>>>
>>> We have had some reproducibly disappointing results with Alexa Fluor 405
>>> tagged secondary antibodies.
>>>
>>> Does anyone have a go-to secondary in this color other than the AF? We
>> are
>>> trying to multiplex as many channels as possible and this is the only one
>>> we
>>> struggle to get good brightness/SNR with.
>>>
>>> Thanks!
>>>
>>> joe
>>>
>>> --------------------------------
>>>
>>> Joe Lebowitz
>>> Ph.D. Candidate
>>> NINDS Pre-doctoral Fellow
>>> Khoshbouei Lab
>>> UF Department of Neuroscience
>>> [phone]
>>>
>>>
>>>

[Debora Olivier (Keller)]

*****
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*****

Hiya,

I've been using dylight 405 on our SP8 confocal (and airyscan) and it works
really well (though I am using it with my strongest primary for instance
Nucleopores).
used in combination with 488 (Alexa or Dylight) and other red secondaries
My mounting medium is based on sorbitol (
https://www.nature.com/articles/s41598-018-32191-x).
I haven't tested it in mountings like prolong et al, I could test it in
vectashield.
I can send a picture if needed.
Cheers

Debora
Debora Olivier, PhD
Research Engineer Microscopy,
Group « Telomeres and Genome Organization »
CBI / LBCMCP
UMR 5088 CNRS/Université Toulouse III
Batiment 4R3 entrée B1
118 Route de Narbonne
31062 Toulouse Cedex, France

telepone: 01 69 33 50 49 (LOB, Palaiseau)
email: [hidden email]
Lab website : https://sites.google.com/yahoo.fr/crabbelab

*NOTE: currently hosted by:*
Laboratory for Optics and Biosciences
INSERM U1182 - CNRS UMR7645
Aisle 2, Level 2 (1st floor)
Ecole Polytechnique
91128 PALAISEAU Cedex


On Fri, May 3, 2019 at 6:07 AM George McNamara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Craig, Joe et al,
>
> as previously noted in another reply, BV421 = Brilliant Violet 421,
> works fine on 405 nm laser. Available from BD Biosciences (acquired the
> manufacturer, Sirigen, for the Brilliants), and BioLegend.
>
> We have a use with a CD## direct labeled primary antibody added to mouse
> leukocytes, imaged live on our Leica SP8 confocal microscope (HyD1, 2nd
> gen hybrid detector), including both time series (100 min) and Z-series.
>
> I note BD Biosciences now has BUVs (excite well ~350 nm), a few BB's
> (excite 488 nm) and one BYG (excite ~561 nm).
>
> I have suggested to a number of users that they move on from DAPI and
> Hoechst to BioLegend's Zombie NIR, excite ok ~640 nm, emission peak ~750
> nm ... BV421-antibody is narrow emission spectrum, bright, and has lots
> of antibodies ... DAPI and Hoechst a broad spectrum, one trick fluors.
>
> enjoy,
>
> George
>
>
> On 5/2/2019 4:47 PM, Craig Brideau wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > What ever happened to Brilliant Violet? I remember a bit of marketing
> about
> > it a while ago, but never got around to trying it myself. Did anyone get
> a
> > chance to give it a go?
> >
> > Craig
> >
> > On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]>
> wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> Hello all,
> >>
> >> We have had some reproducibly disappointing results with Alexa Fluor 405
> >> tagged secondary antibodies.
> >>
> >> Does anyone have a go-to secondary in this color other than the AF? We
> are
> >> trying to multiplex as many channels as possible and this is the only
> one
> >> we
> >> struggle to get good brightness/SNR with.
> >>
> >> Thanks!
> >>
> >> joe
> >>
> >> --------------------------------
> >>
> >> Joe Lebowitz
> >> Ph.D. Candidate
> >> NINDS Pre-doctoral Fellow
> >> Khoshbouei Lab
> >> UF Department of Neuroscience
> >> [phone]
> >>
> >>
> >>
>

[Martin Wessendorf-2]

*****
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Post images on http://www.imgur.com and include the link in your posting.
*****

Hey, Jeff--

As I see it, the main reasons for NOT going with directly conjugated
primary antibodies are (1) the extra time and effort required to perform
the conjugation and clean up the product and (2) the fact that in
principle, conjugation can alter the specificity of an antibody, which
would require repeating its characterization.  However, the latter is a
theoretical issue rather than one that (to my knowledge) has been
documented. Anybody have data on this point?   I've found nothing.

Martin Wessendorf




On 5/2/2019 6:02 PM, Jeff Carmichael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Craig,
>
> You've probably heard some of this from me, and I know that George McNamara
> has also been impressed with and written of the Brilliant Horizon fluors
> from BD (BD owns this from Sirigen purchase).
>
> Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
>
> "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> Hybridization."
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
>
> BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> 25-fold brighter than CFP (depending on who is measuring...)
>
> The problem remains the availability of reagents, as BD has decided that
> their business model is better served by reserving these reagents for their
> custom-conjugated Ab's for flow.....But I believe that BD still entertains
> custom requests for their reagents.
>
> IMHO, directly conjugated, primary Ab's are the best way to leverage these
> fluors because they're so bright that they don't need amplification. This
> removes the problem of species specificity....Why wouldn't one try this???
>
> Here's a link to a presentation that illustrates the efficacy of this
> approach of filling the gap of violet & blue fluors.  Given the headlong
> stampede towards increased multiplexing, I don't understand how these
> fluors could be overlooked:
> https://www.chroma.com/5-channel-fluorescence-imaging-simplified
>
> I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> Technology has no formal relationship with BD Biosciences and receives no
> credit for purchases of related products. Chroma Technology profits only
> from sales of filter sets which are used to image these fluors.
>
> Jeff
>
> *Jeff Carmichael*
>
> *Director of Marketing*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
>
>
> On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> What ever happened to Brilliant Violet? I remember a bit of marketing about
>> it a while ago, but never got around to trying it myself. Did anyone get a
>> chance to give it a go?
>>
>> Craig
>>
>> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]> wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> Post images on http://www.imgur.com and include the link in your
>> posting.
>>> *****
>>>
>>> Hello all,
>>>
>>> We have had some reproducibly disappointing results with Alexa Fluor 405
>>> tagged secondary antibodies.
>>>
>>> Does anyone have a go-to secondary in this color other than the AF? We
>> are
>>> trying to multiplex as many channels as possible and this is the only one
>>> we
>>> struggle to get good brightness/SNR with.
>>>
>>> Thanks!
>>>
>>> joe
>>>
>>> --------------------------------
>>>
>>> Joe Lebowitz
>>> Ph.D. Candidate
>>> NINDS Pre-doctoral Fellow
>>> Khoshbouei Lab
>>> UF Department of Neuroscience
>>> [phone]
>>>
>>>
>>>

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]
My preferred pronouns are "he", "him", and "his"

[Jeffrey Carmichael]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Also, Jackson ImmunoResearch sells some BV421 & BV480 conjugates:
https://www.jacksonimmuno.com/files/JIR-Brilliant-Violet-Conjugates.pdf

Published ext. coef. & quantum yield values from BD Biosciences:

BV421: ɛ (λex) = 2,534,400 ; Φf = 0.70; Excitation maxima = 407nm, Emission
maxima = 421nm

BV480: ɛ (λex) = 1,450,000 ; Φf = 0.69; Excitaiton maxima = 436nm, Emission
maxima = 478nm



Jeff

On Fri, May 3, 2019 at 12:08 AM George McNamara <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Jeff,
>
> Excellent post.
>
> BD business model limitation ... can be dealt with. And the fluorescence
> moicroscopy community would be well served by using as many "flow
> cytometry" compatible reagents as possible.
>
> BD Biosciences and BioLegend each sell BV421 streptavidin conjugates
>
>
> http://www.bdbiosciences.com/us/reagents/research/antibodies-buffers/second-step-reagents/avidinstreptavidin/bv421-streptavidin/p/563259
>
>
> https://www.biolegend.com/en-us/products/brilliant-violet-421-streptavidin-7297
>
> I note that streptavidin has four binding sites for biotin, so may lose
> some efficiency in conjugation = need to optimize amount of BV-SA :
> biotin-mAb ... ideally get 1:1 conjugation, but if a complex forms.
>
> Maybe best counter to BD's business model is to use Fab (or scFv)
> instead of full length IgG. Or VHH nanobodies.
>
> enjoy,
>
> George
>
>
> On 5/2/2019 7:02 PM, Jeff Carmichael wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Craig,
> >
> > You've probably heard some of this from me, and I know that George
> McNamara
> > has also been impressed with and written of the Brilliant Horizon fluors
> > from BD (BD owns this from Sirigen purchase).
> >
> > Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
> >
> > "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> > Hybridization."
> >
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
> >
> > BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> > 25-fold brighter than CFP (depending on who is measuring...)
> >
> > The problem remains the availability of reagents, as BD has decided that
> > their business model is better served by reserving these reagents for
> their
> > custom-conjugated Ab's for flow.....But I believe that BD still
> entertains
> > custom requests for their reagents.
> >
> > IMHO, directly conjugated, primary Ab's are the best way to leverage
> these
> > fluors because they're so bright that they don't need amplification. This
> > removes the problem of species specificity....Why wouldn't one try
> this???
> >
> > Here's a link to a presentation that illustrates the efficacy of this
> > approach of filling the gap of violet & blue fluors.  Given the headlong
> > stampede towards increased multiplexing, I don't understand how these
> > fluors could be overlooked:
> > https://www.chroma.com/5-channel-fluorescence-imaging-simplified
> >
> > I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> > Technology has no formal relationship with BD Biosciences and receives no
> > credit for purchases of related products. Chroma Technology profits only
> > from sales of filter sets which are used to image these fluors.
> >
> > Jeff
> >
> > *Jeff Carmichael*
> >
> > *Director of Marketing*
> >
> > *[hidden email] <[hidden email]> | 802-428-2528*
> >
> >
> >
> > On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> What ever happened to Brilliant Violet? I remember a bit of marketing
> about
> >> it a while ago, but never got around to trying it myself. Did anyone
> get a
> >> chance to give it a go?
> >>
> >> Craig
> >>
> >> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]>
> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> Post images on http://www.imgur.com and include the link in your
> >> posting.
> >>> *****
> >>>
> >>> Hello all,
> >>>
> >>> We have had some reproducibly disappointing results with Alexa Fluor
> 405
> >>> tagged secondary antibodies.
> >>>
> >>> Does anyone have a go-to secondary in this color other than the AF? We
> >> are
> >>> trying to multiplex as many channels as possible and this is the only
> one
> >>> we
> >>> struggle to get good brightness/SNR with.
> >>>
> >>> Thanks!
> >>>
> >>> joe
> >>>
> >>> --------------------------------
> >>>
> >>> Joe Lebowitz
> >>> Ph.D. Candidate
> >>> NINDS Pre-doctoral Fellow
> >>> Khoshbouei Lab
> >>> UF Department of Neuroscience
> >>> [phone]
> >>>
> >>>
> >>>
>

--
 <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>                             

[Jeffrey Carmichael]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Martin,

Yes, I've had the misfortune of ruining perfectly good primary antibodies
in conjugation reactions that resulted in labeled antibodies with excellent
fluorophore:protein ratios, but no remainig antigenicity.

So yes, this is largely hypothetical, but I do know that in the case of the
reference I provided, BD Biosciences did offer assistance with the
conjugation. I'm not sure if the author (David Bates) required the
assistance or not, and I believe he conjugated oligos and not antibodies,
but I believe BD will offer conjugation assistance, as the chemistry with
these polymers can be challenging.

My own take on the extra time is pick your poison....lots of preferred ways
of doing things take more time, and it seems like many folks have a
protocol or two that they are obsessive about. In the end it seems worth
the effort to me. If imaging is the approach a worker has chosen to focus
on to obtain some highly multiplexed data, then it seems that using the
best tools is worth the trouble considering all of the time/effort going
into doing the work at the bench, preparing samples/slides, acquiring the
images, processing the images and then spending the time/effort doing image
analysis. Being able to exploit this violet and blue fluorescence gap in
the spectrum seems like an obvious approach to improve the reach of imaging.

If the data is simply supporting in nature, and not central to the work,
then one probably wouldn't do the extra work. It also seems that in this
age of reproducibility crisis, better tools like these may be part of the
solution.

Jeff

On Fri, May 3, 2019 at 10:38 AM Martin Wessendorf <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hey, Jeff--
>
> As I see it, the main reasons for NOT going with directly conjugated
> primary antibodies are (1) the extra time and effort required to perform
> the conjugation and clean up the product and (2) the fact that in
> principle, conjugation can alter the specificity of an antibody, which
> would require repeating its characterization.  However, the latter is a
> theoretical issue rather than one that (to my knowledge) has been
> documented. Anybody have data on this point?   I've found nothing.
>
> Martin Wessendorf
>
>
>
>
> On 5/2/2019 6:02 PM, Jeff Carmichael wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Craig,
> >
> > You've probably heard some of this from me, and I know that George
> McNamara
> > has also been impressed with and written of the Brilliant Horizon fluors
> > from BD (BD owns this from Sirigen purchase).
> >
> > Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
> >
> > "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> > Hybridization."
> >
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
> >
> > BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> > 25-fold brighter than CFP (depending on who is measuring...)
> >
> > The problem remains the availability of reagents, as BD has decided that
> > their business model is better served by reserving these reagents for
> their
> > custom-conjugated Ab's for flow.....But I believe that BD still
> entertains
> > custom requests for their reagents.
> >
> > IMHO, directly conjugated, primary Ab's are the best way to leverage
> these
> > fluors because they're so bright that they don't need amplification. This
> > removes the problem of species specificity....Why wouldn't one try
> this???
> >
> > Here's a link to a presentation that illustrates the efficacy of this
> > approach of filling the gap of violet & blue fluors.  Given the headlong
> > stampede towards increased multiplexing, I don't understand how these
> > fluors could be overlooked:
> > https://www.chroma.com/5-channel-fluorescence-imaging-simplified
> >
> > I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> > Technology has no formal relationship with BD Biosciences and receives no
> > credit for purchases of related products. Chroma Technology profits only
> > from sales of filter sets which are used to image these fluors.
> >
> > Jeff
> >
> > *Jeff Carmichael*
> >
> > *Director of Marketing*
> >
> > *[hidden email] <[hidden email]> | 802-428-2528*
> >
> >
> >
> > On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> > wrote:
> >
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> Post images on http://www.imgur.com and include the link in your
> posting.
> >> *****
> >>
> >> What ever happened to Brilliant Violet? I remember a bit of marketing
> about
> >> it a while ago, but never got around to trying it myself. Did anyone
> get a
> >> chance to give it a go?
> >>
> >> Craig
> >>
> >> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]>
> wrote:
> >>
> >>> *****
> >>> To join, leave or search the confocal microscopy listserv, go to:
> >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >>> Post images on http://www.imgur.com and include the link in your
> >> posting.
> >>> *****
> >>>
> >>> Hello all,
> >>>
> >>> We have had some reproducibly disappointing results with Alexa Fluor
> 405
> >>> tagged secondary antibodies.
> >>>
> >>> Does anyone have a go-to secondary in this color other than the AF? We
> >> are
> >>> trying to multiplex as many channels as possible and this is the only
> one
> >>> we
> >>> struggle to get good brightness/SNR with.
> >>>
> >>> Thanks!
> >>>
> >>> joe
> >>>
> >>> --------------------------------
> >>>
> >>> Joe Lebowitz
> >>> Ph.D. Candidate
> >>> NINDS Pre-doctoral Fellow
> >>> Khoshbouei Lab
> >>> UF Department of Neuroscience
> >>> [phone]
> >>>
> >>>
> >>>
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
> My preferred pronouns are "he", "him", and "his"
>

--
 <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>                             

[Craig Brideau]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Thanks for the interesting discussion. Interesting to note the potential
change in binding efficacy when you attach the fluorophore in the case of a
polymer. How does this compare to the coating chemistry of quantum dots?

Craig

On Fri, May 3, 2019 at 10:07 AM Jeff Carmichael <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Martin,
>
> Yes, I've had the misfortune of ruining perfectly good primary antibodies
> in conjugation reactions that resulted in labeled antibodies with excellent
> fluorophore:protein ratios, but no remainig antigenicity.
>
> So yes, this is largely hypothetical, but I do know that in the case of the
> reference I provided, BD Biosciences did offer assistance with the
> conjugation. I'm not sure if the author (David Bates) required the
> assistance or not, and I believe he conjugated oligos and not antibodies,
> but I believe BD will offer conjugation assistance, as the chemistry with
> these polymers can be challenging.
>
> My own take on the extra time is pick your poison....lots of preferred ways
> of doing things take more time, and it seems like many folks have a
> protocol or two that they are obsessive about. In the end it seems worth
> the effort to me. If imaging is the approach a worker has chosen to focus
> on to obtain some highly multiplexed data, then it seems that using the
> best tools is worth the trouble considering all of the time/effort going
> into doing the work at the bench, preparing samples/slides, acquiring the
> images, processing the images and then spending the time/effort doing image
> analysis. Being able to exploit this violet and blue fluorescence gap in
> the spectrum seems like an obvious approach to improve the reach of
> imaging.
>
> If the data is simply supporting in nature, and not central to the work,
> then one probably wouldn't do the extra work. It also seems that in this
> age of reproducibility crisis, better tools like these may be part of the
> solution.
>
> Jeff
>
> On Fri, May 3, 2019 at 10:38 AM Martin Wessendorf <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Hey, Jeff--
> >
> > As I see it, the main reasons for NOT going with directly conjugated
> > primary antibodies are (1) the extra time and effort required to perform
> > the conjugation and clean up the product and (2) the fact that in
> > principle, conjugation can alter the specificity of an antibody, which
> > would require repeating its characterization.  However, the latter is a
> > theoretical issue rather than one that (to my knowledge) has been
> > documented. Anybody have data on this point?   I've found nothing.
> >
> > Martin Wessendorf
> >
> >
> >
> >
> > On 5/2/2019 6:02 PM, Jeff Carmichael wrote:
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Craig,
> > >
> > > You've probably heard some of this from me, and I know that George
> > McNamara
> > > has also been impressed with and written of the Brilliant Horizon
> fluors
> > > from BD (BD owns this from Sirigen purchase).
> > >
> > > Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
> > >
> > > "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> > > Hybridization."
> > >
> >
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
> > >
> > > BV421 is approx. 20-40-fold brighter than AF405. BV480 is also approx.
> > > 25-fold brighter than CFP (depending on who is measuring...)
> > >
> > > The problem remains the availability of reagents, as BD has decided
> that
> > > their business model is better served by reserving these reagents for
> > their
> > > custom-conjugated Ab's for flow.....But I believe that BD still
> > entertains
> > > custom requests for their reagents.
> > >
> > > IMHO, directly conjugated, primary Ab's are the best way to leverage
> > these
> > > fluors because they're so bright that they don't need amplification.
> This
> > > removes the problem of species specificity....Why wouldn't one try
> > this???
> > >
> > > Here's a link to a presentation that illustrates the efficacy of this
> > > approach of filling the gap of violet & blue fluors.  Given the
> headlong
> > > stampede towards increased multiplexing, I don't understand how these
> > > fluors could be overlooked:
> > > https://www.chroma.com/5-channel-fluorescence-imaging-simplified
> > >
> > > I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> > > Technology has no formal relationship with BD Biosciences and receives
> no
> > > credit for purchases of related products. Chroma Technology profits
> only
> > > from sales of filter sets which are used to image these fluors.
> > >
> > > Jeff
> > >
> > > *Jeff Carmichael*
> > >
> > > *Director of Marketing*
> > >
> > > *[hidden email] <[hidden email]> | 802-428-2528*
> > >
> > >
> > >
> > > On Thu, May 2, 2019 at 4:47 PM Craig Brideau <[hidden email]>
> > > wrote:
> > >
> > >> *****
> > >> To join, leave or search the confocal microscopy listserv, go to:
> > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> Post images on http://www.imgur.com and include the link in your
> > posting.
> > >> *****
> > >>
> > >> What ever happened to Brilliant Violet? I remember a bit of marketing
> > about
> > >> it a while ago, but never got around to trying it myself. Did anyone
> > get a
> > >> chance to give it a go?
> > >>
> > >> Craig
> > >>
> > >> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]>
> > wrote:
> > >>
> > >>> *****
> > >>> To join, leave or search the confocal microscopy listserv, go to:
> > >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >>> Post images on http://www.imgur.com and include the link in your
> > >> posting.
> > >>> *****
> > >>>
> > >>> Hello all,
> > >>>
> > >>> We have had some reproducibly disappointing results with Alexa Fluor
> > 405
> > >>> tagged secondary antibodies.
> > >>>
> > >>> Does anyone have a go-to secondary in this color other than the AF?
> We
> > >> are
> > >>> trying to multiplex as many channels as possible and this is the only
> > one
> > >>> we
> > >>> struggle to get good brightness/SNR with.
> > >>>
> > >>> Thanks!
> > >>>
> > >>> joe
> > >>>
> > >>> --------------------------------
> > >>>
> > >>> Joe Lebowitz
> > >>> Ph.D. Candidate
> > >>> NINDS Pre-doctoral Fellow
> > >>> Khoshbouei Lab
> > >>> UF Department of Neuroscience
> > >>> [phone]
> > >>>
> > >>>
> > >>>
> >
> > --
> > Martin Wessendorf, Ph.D.                   office: [phone]
> > Assoc Prof, Dept Neuroscience                 lab: [phone]
> > University of Minnesota             Preferred FAX: [phone]
> > 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> > Minneapolis, MN  55455                    e-mail: [hidden email]
> > My preferred pronouns are "he", "him", and "his"
> >
>
> --
>  <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
> *an employee owned
> company*
> 10 Imtec Lane, Bellows Falls, Vermont 05101 USA
> 800-824-7662 |
> FAX: [phone]
> www.chroma.com <https://www.chroma.com/> |
> [hidden email] <mailto:[hidden email]>
>

[Jeffrey Carmichael]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

So interestingly, not just for large polymers, but this is a potential
problem when chemically conjugating anything to an antibody. My unfortunate
experience was with Alexa Fluors and Oregon Green 488, small organic
molecules. Here's a link to a paper in the Biophysical Journal (2018)
describing the alteration of antigen binding following antibody conjugation
to one of two different AlexaFluors:
https://www.cell.com/biophysj/pdf/S0006-3495(17)35091-9.pdf

I suspect that there are some additional concerns when it involves large
polymers, but in general it seems to be a steric type of conformational
hindrance, not necessarily related to the particular chemical moiety, like
a conductive polymer vs. a ZnS QDot shell vs. small organic fluorophore vs.
the chemical linker used in the conjugation.

Jeff



On Fri, May 3, 2019 at 12:34 PM Craig Brideau <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for the interesting discussion. Interesting to note the potential
> change in binding efficacy when you attach the fluorophore in the case of a
> polymer. How does this compare to the coating chemistry of quantum dots?
>
> Craig
>
> On Fri, May 3, 2019 at 10:07 AM Jeff Carmichael <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > Post images on http://www.imgur.com and include the link in your
> posting.
> > *****
> >
> > Martin,
> >
> > Yes, I've had the misfortune of ruining perfectly good primary antibodies
> > in conjugation reactions that resulted in labeled antibodies with
> excellent
> > fluorophore:protein ratios, but no remainig antigenicity.
> >
> > So yes, this is largely hypothetical, but I do know that in the case of
> the
> > reference I provided, BD Biosciences did offer assistance with the
> > conjugation. I'm not sure if the author (David Bates) required the
> > assistance or not, and I believe he conjugated oligos and not antibodies,
> > but I believe BD will offer conjugation assistance, as the chemistry with
> > these polymers can be challenging.
> >
> > My own take on the extra time is pick your poison....lots of preferred
> ways
> > of doing things take more time, and it seems like many folks have a
> > protocol or two that they are obsessive about. In the end it seems worth
> > the effort to me. If imaging is the approach a worker has chosen to focus
> > on to obtain some highly multiplexed data, then it seems that using the
> > best tools is worth the trouble considering all of the time/effort going
> > into doing the work at the bench, preparing samples/slides, acquiring the
> > images, processing the images and then spending the time/effort doing
> image
> > analysis. Being able to exploit this violet and blue fluorescence gap in
> > the spectrum seems like an obvious approach to improve the reach of
> > imaging.
> >
> > If the data is simply supporting in nature, and not central to the work,
> > then one probably wouldn't do the extra work. It also seems that in this
> > age of reproducibility crisis, better tools like these may be part of the
> > solution.
> >
> > Jeff
> >
> > On Fri, May 3, 2019 at 10:38 AM Martin Wessendorf <[hidden email]>
> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > Post images on http://www.imgur.com and include the link in your
> > posting.
> > > *****
> > >
> > > Hey, Jeff--
> > >
> > > As I see it, the main reasons for NOT going with directly conjugated
> > > primary antibodies are (1) the extra time and effort required to
> perform
> > > the conjugation and clean up the product and (2) the fact that in
> > > principle, conjugation can alter the specificity of an antibody, which
> > > would require repeating its characterization.  However, the latter is a
> > > theoretical issue rather than one that (to my knowledge) has been
> > > documented. Anybody have data on this point?   I've found nothing.
> > >
> > > Martin Wessendorf
> > >
> > >
> > >
> > >
> > > On 5/2/2019 6:02 PM, Jeff Carmichael wrote:
> > > > *****
> > > > To join, leave or search the confocal microscopy listserv, go to:
> > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > > Post images on http://www.imgur.com and include the link in your
> > > posting.
> > > > *****
> > > >
> > > > Craig,
> > > >
> > > > You've probably heard some of this from me, and I know that George
> > > McNamara
> > > > has also been impressed with and written of the Brilliant Horizon
> > fluors
> > > > from BD (BD owns this from Sirigen purchase).
> > > >
> > > > Here's a reference using Brilliant Violet 421 & Brilliant Violet 480:
> > > >
> > > > "Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ
> > > > Hybridization."
> > > >
> > >
> >
> https://www.researchgate.net/publication/319289637_Multilocus_Imaging_of_the_E_coli_Chromosome_by_Fluorescent_In_Situ_Hybridization
> > > >
> > > > BV421 is approx. 20-40-fold brighter than AF405. BV480 is also
> approx.
> > > > 25-fold brighter than CFP (depending on who is measuring...)
> > > >
> > > > The problem remains the availability of reagents, as BD has decided
> > that
> > > > their business model is better served by reserving these reagents for
> > > their
> > > > custom-conjugated Ab's for flow.....But I believe that BD still
> > > entertains
> > > > custom requests for their reagents.
> > > >
> > > > IMHO, directly conjugated, primary Ab's are the best way to leverage
> > > these
> > > > fluors because they're so bright that they don't need amplification.
> > This
> > > > removes the problem of species specificity....Why wouldn't one try
> > > this???
> > > >
> > > > Here's a link to a presentation that illustrates the efficacy of this
> > > > approach of filling the gap of violet & blue fluors.  Given the
> > headlong
> > > > stampede towards increased multiplexing, I don't understand how these
> > > > fluors could be overlooked:
> > > > https://www.chroma.com/5-channel-fluorescence-imaging-simplified
> > > >
> > > > I hesitate using the "COMMERCIAL RESPONSE" qualifier, because Chroma
> > > > Technology has no formal relationship with BD Biosciences and
> receives
> > no
> > > > credit for purchases of related products. Chroma Technology profits
> > only
> > > > from sales of filter sets which are used to image these fluors.
> > > >
> > > > Jeff
> > > >
> > > > *Jeff Carmichael*
> > > >
> > > > *Director of Marketing*
> > > >
> > > > *[hidden email] <[hidden email]> | 802-428-2528*
> > > >
> > > >
> > > >
> > > > On Thu, May 2, 2019 at 4:47 PM Craig Brideau <
> [hidden email]>
> > > > wrote:
> > > >
> > > >> *****
> > > >> To join, leave or search the confocal microscopy listserv, go to:
> > > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > >> Post images on http://www.imgur.com and include the link in your
> > > posting.
> > > >> *****
> > > >>
> > > >> What ever happened to Brilliant Violet? I remember a bit of
> marketing
> > > about
> > > >> it a while ago, but never got around to trying it myself. Did anyone
> > > get a
> > > >> chance to give it a go?
> > > >>
> > > >> Craig
> > > >>
> > > >> On Thu, May 2, 2019 at 2:27 PM Joe Lebowitz <[hidden email]>
> > > wrote:
> > > >>
> > > >>> *****
> > > >>> To join, leave or search the confocal microscopy listserv, go to:
> > > >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > >>> Post images on http://www.imgur.com and include the link in your
> > > >> posting.
> > > >>> *****
> > > >>>
> > > >>> Hello all,
> > > >>>
> > > >>> We have had some reproducibly disappointing results with Alexa
> Fluor
> > > 405
> > > >>> tagged secondary antibodies.
> > > >>>
> > > >>> Does anyone have a go-to secondary in this color other than the AF?
> > We
> > > >> are
> > > >>> trying to multiplex as many channels as possible and this is the
> only
> > > one
> > > >>> we
> > > >>> struggle to get good brightness/SNR with.
> > > >>>
> > > >>> Thanks!
> > > >>>
> > > >>> joe
> > > >>>
> > > >>> --------------------------------
> > > >>>
> > > >>> Joe Lebowitz
> > > >>> Ph.D. Candidate
> > > >>> NINDS Pre-doctoral Fellow
> > > >>> Khoshbouei Lab
> > > >>> UF Department of Neuroscience
> > > >>> [phone]
> > > >>>
> > > >>>
> > > >>>
> > >
> > > --
> > > Martin Wessendorf, Ph.D.                   office: [phone]
> > > Assoc Prof, Dept Neuroscience                 lab: [phone]
> > > University of Minnesota             Preferred FAX: [phone]
> > > 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> > > Minneapolis, MN  55455                    e-mail: [hidden email]
> > > My preferred pronouns are "he", "him", and "his"
> > >
> >
> > --
> >  <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
> > *an employee owned
> > company*
> > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA
> > 800-824-7662 |
> > FAX: [phone]
> > www.chroma.com <https://www.chroma.com/> |
> > [hidden email] <mailto:[hidden email]>
> >
>

--
 <https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: [phone]
www.chroma.com <https://www.chroma.com/> | 
[hidden email] <mailto:[hidden email]>