Confocal Microscopy List

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

Classic

List

Threaded

3 messages Options

Kilgore, Jason A.

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

*****
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.
*****

** Vendor Reply **

Hi, Doug,

Blue dyes in general have a lower extinction coefficient, and the lower absorbance results in a dimmer emission. Alexa Fluor 350 and 405 are great options, but you may still need to amplify over background.

To that end, may I suggest that you try a biotinylated probe followed by the streptavidin form of AF350 or AF405?

Or, you can amplify further by using an HRP conjugate and following with an AF350 tyramide.

Please feel free to contact me offline if you'd like some more help on these options or catalog numbers.

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific

1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Moulding, Dale
Sent: Monday, August 20, 2018 11:02 PM
To: [hidden email]
Subject: Re: recommendation for a "blue" secondary fluorochrome

CAUTION: This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Cromey, Douglas W - (dcromey) <[hidden email]>
Sent: 21 August 2018 00:06:28
To: [hidden email]
Subject: recommendation for a "blue" secondary fluorochrome

*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=QMhO69omek1WNOABbr_6NSbnMrAERcZTor5ylr9ywFk&e=
LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=QMhO69omek1WNOABbr_6NSbnMrAERcZTor5ylr9ywFk&e=>
lists.umn.edu
[hidden email]: listserv archives. confocalmicroscopy

Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=y4Q4EHuTB12c0g1FcMvKEKOfyV6NOnyyVVc5Wkkedek&e= and include the link in your posting.
*****

Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.

I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.

The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?

Thanks,
Doug

------------------------------------------------------------------------------------------
Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA

office: LSN 463 email: [hidden email]<mailto:[hidden email]>
voice: 520-626-2824 fax: 520-626-2097

https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopy.arizona.edu_learn_microscopy-2Dimaging-2Dresources-2Dwww&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=zlrzolD0U1H_GeyE5y-ypbZetO_vGw9p_17mN3LTd9Y&e=
Home of: "Microscopy and Imaging Resources on the WWW"

UA Microscopy Alliance - https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopy.arizona.edu&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=iOrsCj13ORNkyOfJH3HsZC0YddTDhKBF6IoyI8fWJgY&e=<https://urldefense.proofpoint.com/v2/url?u=http-3A__microscopy.arizona.edu_&d=DwIFAw&c=q6k2DsTcEGCcCb_WtVSz6hhIl8hvYssy7sH8ZwfbbKU&r=MVp-2yJ1A-yQtCbZ-DE9xd0W6E7srQQpV-yioYjTLyQ&m=GyNL_oO-pPQ-tnnmvV1UL295qKZGQfBbIjPXeUr-M2A&s=iwD73SMVPqjK6QJUxOpm4g4qjYjYXlaoKRbIMaxBUVU&e=>

Jeffrey Carmichael

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

*****
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.
*****

Jason,

I agree that most of the Alexa Fluors are excellent fluorophores, but I
disagree that either AF350 or AF405 are good options. They're not very
photostable and are dim. I was never able to get satisfactory results with
them unless labeling something like actin with phalloidin.

In contrast, the extinction coefficient of BV421 is 2,534,400 with a QY =
0.70. Extinction coefficient of BV480 is 1,450,000, QY = 0.69. I don't know
if the photostability has been quantified, but it's extreme.

Again, the potential drawback with these is chemistry. However, please see
the reference below. David Bates at Baylor College of Medicine published
results of 5 color FISH in bacteria using BV480 as the blue/cyan channel
and separated it from AF488:

https://link.springer.com/protocol/10.1007%2F978-1-4939-7098-8_16

From a figure in the paper:

*Fig. 3 *Five-color FISH with DAPI in *E. coli*. Phase contrast with DAPI
fluorescence image (left panel). Combined 5-color FISH image (right panel). *E.
coli *cells exponentially growing in minimal media (containing ≤ 2
chromosomes) were hybridized with 3 kb probes targeted to 5 equally spaced
(~500 kb) sites on the *E. coli *chromosome. Probes were labeled by nick
translation with the following nucleotide base conjugates: digoxigenin-dUTP
secondarily detected with anti-digoxigenin BV480 (blue), AF488-dUTP
(green), AF546-dUTP (yellow), AF594-dUTP (red), and AF647-dUTP (magenta).
Brightness and contrast of color channels were independently adjusted and
merged into a composite image in Axiovision. Bar is 5 μm.
They were unable to use nick translation with the BV480, and instead used
an anti-DIG secondary conjugated to BV480. Directly conjugated oligos are
another option in FISH.

Admittedly, it's very easy access to single bacteria in terms of
penetration/diffusion of a large probe, and not challenging as with tissue.

Jeff

*Jeff Carmichael*
*Product & Technical Marketing Manager*
*[hidden email] <[hidden email]> | 802-428-2528*

** Vendor Reply **

Hi, Doug,

Blue dyes in general have a lower extinction coefficient, and the lower
absorbance results in a dimmer emission. Alexa Fluor 350 and 405 are great
options, but you may still need to amplify over background.

To that end, may I suggest that you try a biotinylated probe followed by
the streptavidin form of AF350 or AF405?

Or, you can amplify further by using an HRP conjugate and following with an
AF350 tyramide.

Please feel free to contact me offline if you'd like some more help on
these options or catalog numbers.

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific

1-800-955-6288 then option 4, then option 3, then option 2.
Or dial direct at +1 541 335 0353
[hidden email]

This communication is intended solely for the individual/entity to whom it
is addressed. It may contain confidential or legally privileged
information. Any unauthorized disclosure or copying is prohibited and may
be unlawful. If you have received this communication in error, please
notify the sender immediately and delete it from your system.

*****

This is correct.

My understanding is that BV421, BV480 & BB515 are all "native", primary
fluors without "acceptors" and the additional, attendant fluorescence
wavelengths. I've been out of the loop lately, but additional "primary"
Brillinat Horizon fluors are likely to be introduced by BD Biosciences.

What is often overlooked with these fluors is that they are so bright and
photostable that their real potential value is to use them for directly
labelled, primary antibodies, without need for amplification and the issues
that can arise from using secondary antibodies. I think it remains to be
seen how useful they might ultimately be as primary probes.

Perhaps the problems associated with direct primary antibody conjugation
such as:

- maintaining the antigenicity of the antibody
- permeability into tissue due to size

might be too difficult to overcome, but because of their brightness &
photostability, it seems worth the effort to try for those folks who are
interested in developing new/improved methodologies.

Jeff

*Jeff Carmichael*
*Product & Technical Marketing Manager*

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

* <[hidden email]>*

On Tue, Aug 21, 2018 at 8:32 AM, Adrian Smith <[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.
> *****
>
> Most of the longer wavelength Brilliant Violet reagents are tandems, ie a
> BV-polymer backbone as a donor with a second molecule as acceptor - hence
> the excitation at 561/594/633.
>
> Since Sirigin was purchased by Becton Dickinson they have come out with
> additional base polymers - Brilliant UV, Brilliant Blue, Brilliant
> Yellow/Green (may not be commercially available yet). Generally in each
> series the first reagent is a straight polymer and subsequent ones are
> tandems. As I know only BD is producing these and the focus has been on
> reagents for flow cytometry.
>
> http://www.bdbiosciences.com/anz/go/brilliant/
>
> (this is the ANZ site - US site is down for me at the moment)
>
> Regards,
>
> Adrian
>
>
>
> On Tue, 21 Aug 2018 at 22:15, Moulding, Dale <[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.
> > *****
> >
> > I've tried the BV dyes. I may have got the wrong impression about them,
> > but thought they were relatively large? Certainly for whole mount
> staining
> > I thought they didn’t penetrate tissue very well.
> > I also found that while BV421 was useful, the longer wavelength BV dyes
> > when used on monolayers gave quite considerable fluorescence when

excited
> > at 561, 594 or 633 nm, making them less useful than we had hoped for
> > multichannel imaging without unmixing.
> > I'd be very interested to hear if I'm mistaken, and they are in fact
> small
> > molecules that can penetrate well into whole mounts. If so we would
> > certainly switch to BV421 instead of Alexa405. Empirically we do find
> > alexa405 is not particularly bright, but generally find it to be as
about

> > as photostable as Alexa568.
> >
> > Cheers
> >
> > Dale
> >
> > -----Original Message-----
> > From: Confocal Microscopy List <[hidden email]> On
> > Behalf Of Jeff Carmichael
> > Sent: 21 August 2018 12:31
> > To: [hidden email]
> > Subject: Re: recommendation for a "blue" secondary fluorochrome
> COMMERCIAL
> > RESPONSE
> >
> > *****
> > 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.
> > *****
> >
> > This has been addressed more than once in the listserv, but below are
> some
> > resources to look into the BrilliantViolet fluorophores which are much
> more
> > than an order of magnitude brighter than AF405. You could potentially

add

> > two additional channels in this spectral "dead zone".
> >
> > Chroma Technology has no commercial interest in these fluorophores, only
> > in the filter sets we've recommended for detecting them.
> >
> >
> > https://www.jacksonimmuno.com/technical/products/conjugate-
> selection/brilliant-violet
> > http://bit.ly/ChromaTechnology2MtEdC9
> > http://bit.ly/bdbiosciences2MEed69
> >
> > Good luck and please share your results!
> > Jeff
> >
> > *Jeff Carmichael*
> > *Product & Technical Marketing Manager*
> >
> > *[hidden email] <[hidden email]> | 802-428-2528*
> >
> > * <[hidden email]>*
> >
> > On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) <
> > [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.
> > > *****
> > >
> > > Is there a better or preferred "blue" fluorochrome? Mostly this is a
> > > widefield fluorescence question (using a DAPI cube), but there is the
> > > possibility for using a confocal with a 405nm laser line.
> > >
> > > I'm working with a group that is trying to work around a frustrating
> > > autofluorescence issue in liver tissue. What we ended up doing on an
> > > earlier project was not using any secondaries that fluoresced in the
> > > green channel (no fitc/AF488/GFP) and instead used the green channel
> > > on our widefield microscope as a control, ratioing this image so we
> > > could subtract it out of the other channels to remove some of the
> > > broad autofluorescent background. This worked nicely. In the published
> > > images we showed nuclei
> > > (blue) and two specific stains using AF568 and AF647, with reduced
> > > background autofluorescence.
> > >
> > > The PI now wants to have labelling with three specific fluorochromes,
> > > and I am advocating using the green channel as the generic "everything
> > > lights up" channel for context, so we can hopefully skip the Hoechst
> > 3342 or DAPI.
> > > Since we won't be using DNA dyes, is there a good blue fluorochrome
> > > that can be used as a secondary for specific labelling of a marker
> > > found on an intracellular organelle (other than the nucleus)?
> > >
> > > Thanks,
> > > Doug
> > >
> > > ------------------------------------------------------------
> > > ------------------------------
> > > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
> > > Cellular & Molecular Medicine, University of Arizona
> > > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
> > >
> > > office: LSN 463 email: [hidden email]<mailto:
> > > [hidden email]>
> > > voice: 520-626-2824 fax: 520-626-2097
> > >
> > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
> > > Home of: "Microscopy and Imaging Resources on the WWW"
> > >
> > > UA Microscopy Alliance - http://microscopy.arizona.edu<
> > > http://microscopy.arizona.edu/>
> > >
> >
> > --
> > <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned
> > company*
> > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA
> > 800-824-7662 |
> > FAX: 802-428-2525
> > www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:
> > [hidden email]>
> >
>
> --
> <https://www.centenary.org.au>
>

--
<https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: 802-428-2525
www.chroma.com <https://www.chroma.com/> |
[hidden email] <mailto:[hidden email]>

---------- Forwarded message ----------
From: Jeff Carmichael <[hidden email]>
To: Confocal Microscopy List <[hidden email]>
Cc:
Bcc:
Date: Tue, 21 Aug 2018 09:22:46 -0400
Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL
RESPONSE
*****
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.
*****

This is correct.

My understanding is that BV421, BV480 & BB515 are all "native", primary
fluors without "acceptors" and the additional, attendant fluorescence
wavelengths. I've been out of the loop lately, but additional "primary"
Brillinat Horizon fluors are likely to be introduced by BD Biosciences.

What is often overlooked with these fluors is that they are so bright and
photostable that their real potential value is to use them for directly
labelled, primary antibodies, without need for amplification and the issues
that can arise from using secondary antibodies. I think it remains to be
seen how useful they might ultimately be as primary probes.

Perhaps the problems associated with direct primary antibody conjugation
such as:

- maintaining the antigenicity of the antibody
- permeability into tissue due to size

might be too difficult to overcome, but because of their brightness &
photostability, it seems worth the effort to try for those folks who are
interested in developing new/improved methodologies.

Jeff

*Jeff Carmichael*
*Product & Technical Marketing Manager*

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

* <[hidden email]>*

On Tue, Aug 21, 2018 at 8:32 AM, Adrian Smith <[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.
> *****
>
> Most of the longer wavelength Brilliant Violet reagents are tandems, ie a
> BV-polymer backbone as a donor with a second molecule as acceptor - hence
> the excitation at 561/594/633.
>
> Since Sirigin was purchased by Becton Dickinson they have come out with
> additional base polymers - Brilliant UV, Brilliant Blue, Brilliant
> Yellow/Green (may not be commercially available yet). Generally in each
> series the first reagent is a straight polymer and subsequent ones are
> tandems. As I know only BD is producing these and the focus has been on
> reagents for flow cytometry.
>
> http://www.bdbiosciences.com/anz/go/brilliant/
>
> (this is the ANZ site - US site is down for me at the moment)
>
> Regards,
>
> Adrian
>
>
>
> On Tue, 21 Aug 2018 at 22:15, Moulding, Dale <[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.
> > *****
> >
> > I've tried the BV dyes. I may have got the wrong impression about them,
> > but thought they were relatively large? Certainly for whole mount
> staining
> > I thought they didn’t penetrate tissue very well.
> > I also found that while BV421 was useful, the longer wavelength BV dyes
> > when used on monolayers gave quite considerable fluorescence when

add

--
<https://www.chroma.com/>CHROMA TECHNOLOGY CORP®
*an employee owned
company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 |
FAX: 802-428-2525
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: 802-428-2525
www.chroma.com <https://www.chroma.com/> |
[hidden email] <mailto:[hidden email]>

George McNamara

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

*****
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 Doug, Jeff, et al,

I like Jeff's suggestion of Brilliant(s).

Another way to go is fluorescent tyramide signal amplification (might
help with AF350, AF430, etc), or for extreme, TSA-hapten and detect with
Brilliant(s).

7plex TSA was published in 2000:

http://journals.sagepub.com/doi/10.1177/002215540004800509

https://www.ncbi.nlm.nih.gov/pubmed/?term=tsurui+2000+spectral

Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T. Seven-color
fluorescence imaging of tissue samples based on Fourier spectroscopy and singular
value decomposition. J Histochem Cytochem. 2000 May;48(5):653-62. PubMed PMID:
10769049.

PerkinElmer now sells 7plex Opal, as well as 4plex Opal.

Two papers on N-methylated luminol report on a reagent that may be 10x
better than fluorescent tyramide (could someone please email me offline
about why NADH works with the luminol, 2nd abstract):

https://www.ncbi.nlm.nih.gov/pubmed/26356088

https://www.ncbi.nlm.nih.gov/pubmed/28009088

ACS Chem Biol. 2015 Nov 20;10(11):2633-40. doi: 10.1021/acschembio.5b00440. Epub
2015 Sep 18.

Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated Luminol
Derivatives.

Sato S(1), Nakamura K(1), Nakamura H(1).

Author information:
(1)Chemical Resources Laboratory, Tokyo Institute of Technology , Yokohama
226-8503, Japan.

Tyrosine-specific chemical modification was achieved using in situ
hemin-activated luminol derivatives. Tyrosine residues in peptide and protein
were modified effectively with N-methylated luminol derivatives under oxidative
conditions in the presence of hemin and H2O2. Both single and double
modifications of the tyrosine residue occurred in the reaction of angiotensin II
with N-methylated luminol derivative 9. Tyrosine-specific chemical modification
of the model protein bovine serum albumin (BSA) revealed that the surface-exposed
tyrosine residues were selectively modified with 9. We succeeded in the
functionalization of several proteins using azide-conjugated compound 18 using
alkyne-conjugated probes by copper(I)-catalyzed azide-alkyne cycloaddition
(CuAAC) or dibenzocyclooctyne (DBCO)-mediated copper-free click chemistry. This
tyrosine-specific modification was orthogonal to conventional lysine modification
by N-hydroxysuccinimide (NHS) ester, and dual functionalization by fluorescence
modification of tyrosine residues and PEG modification of lysine residues was
achieved without affecting the modification efficiency.

DOI: 10.1021/acschembio.5b00440
PMID: 26356088

Chembiochem. 2017 Mar 2;18(5):475-478. doi: 10.1002/cbic.201600649. Epub 2017 Jan
23.

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

Sato S(1), Nakamura K(1), Nakamura H(1).

Author information:
(1)Laboratory for Chemistry and Life Science, Institute of Innovative Research,
Tokyo Institute of Technology, Yokohama, 226-8503, Japan.

The efficiency of protein chemical modification on tyrosine residues with
N-methylluminol derivatives was drastically improved by using horseradish
peroxidase (HRP). In the previous method, based on the use of hemin and H2 O2 ,
oxidative side reactions such as cysteine oxidation were problematic for
functionalization of proteins selectively on tyrosine residues. Oxidative
activation of N-methylluminol derivatives with a minimum amount of H2 O2
prevented the occurrence of oxidative side reactions under HRP-catalyzed
conditions. As probes for HRP-catalyzed protein modification, N-methylluminol
derivatives showed much higher efficiency than tyramide without inducing
oligomerization of probe molecules. Tyrosine modification also proceeded in the
presence of β-nicotinamide adenine dinucleotide (NADH, H2 O2 -free conditions).

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI: 10.1002/cbic.201600649
PMID: 28009088

For HRP, consider HRP80

https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s120.html

"The estimated average number of HRP monomer molecules in SA-PolyHRP20
conjugate is 100 (20 X 5), in SA-PolyHRP40 - 200 (40 X 5) and in
SA-PolyHRP80 - 400 (80 X 5). Thus, PolyHRP brings in reaction with
substrate development system much larger number of enzyme label
molecules (per one bound analyte molecule) than conventional conjugates do."

My expectation is most of the fluorescent tyramides or luminols will
bind to the HRPs ... as long as you don't zap them off with standard
Opal protocol

My former colleagues at UMiami identified PeroxAbolish as an excellent
way to kill HRP without removal.

https://biocare.net/product/peroxabolish/

Image quality was a lot better than the published image:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777543/figure/F2/

1. Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information:
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747
PMCID: PMC3777543
PMID: 21929847

enjoy,

George

On 8/21/2018 1:38 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.
> *****
>
> Jason,
>
> I agree that most of the Alexa Fluors are excellent fluorophores, but I
> disagree that either AF350 or AF405 are good options. They're not very
> photostable and are dim. I was never able to get satisfactory results with
> them unless labeling something like actin with phalloidin.
>
> In contrast, the extinction coefficient of BV421 is 2,534,400 with a QY =
> 0.70. Extinction coefficient of BV480 is 1,450,000, QY = 0.69. I don't know
> if the photostability has been quantified, but it's extreme.
>
> Again, the potential drawback with these is chemistry. However, please see
> the reference below. David Bates at Baylor College of Medicine published
> results of 5 color FISH in bacteria using BV480 as the blue/cyan channel
> and separated it from AF488:
>
> https://link.springer.com/protocol/10.1007%2F978-1-4939-7098-8_16
>
> From a figure in the paper:
>
> *Fig. 3 *Five-color FISH with DAPI in *E. coli*. Phase contrast with DAPI
> fluorescence image (left panel). Combined 5-color FISH image (right panel). *E.
> coli *cells exponentially growing in minimal media (containing ≤ 2
> chromosomes) were hybridized with 3 kb probes targeted to 5 equally spaced
> (~500 kb) sites on the *E. coli *chromosome. Probes were labeled by nick
> translation with the following nucleotide base conjugates: digoxigenin-dUTP
> secondarily detected with anti-digoxigenin BV480 (blue), AF488-dUTP
> (green), AF546-dUTP (yellow), AF594-dUTP (red), and AF647-dUTP (magenta).
> Brightness and contrast of color channels were independently adjusted and
> merged into a composite image in Axiovision. Bar is 5 μm.
> They were unable to use nick translation with the BV480, and instead used
> an anti-DIG secondary conjugated to BV480. Directly conjugated oligos are
> another option in FISH.
>
> Admittedly, it's very easy access to single bacteria in terms of
> penetration/diffusion of a large probe, and not challenging as with tissue.
>
> Jeff
>
> *Jeff Carmichael*
> *Product & Technical Marketing Manager*
> *[hidden email] <[hidden email]> | 802-428-2528*
>
>
>
>
> ** Vendor Reply **
>
> Hi, Doug,
>
> Blue dyes in general have a lower extinction coefficient, and the lower
> absorbance results in a dimmer emission. Alexa Fluor 350 and 405 are great
> options, but you may still need to amplify over background.
>
> To that end, may I suggest that you try a biotinylated probe followed by
> the streptavidin form of AF350 or AF405?
>
> Or, you can amplify further by using an HRP conjugate and following with an
> AF350 tyramide.
>
> Please feel free to contact me offline if you'd like some more help on
> these options or catalog numbers.
>
> Jason
>
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes / EVOS Tech Support
> Thermo Fisher Scientific
>
> 1-800-955-6288 then option 4, then option 3, then option 2.
> Or dial direct at +1 541 335 0353
> [hidden email]
>
> This communication is intended solely for the individual/entity to whom it
> is addressed. It may contain confidential or legally privileged
> information. Any unauthorized disclosure or copying is prohibited and may
> be unlawful. If you have received this communication in error, please
> notify the sender immediately and delete it from your system.
>
> *****
>
> This is correct.
>
> My understanding is that BV421, BV480 & BB515 are all "native", primary
> fluors without "acceptors" and the additional, attendant fluorescence
> wavelengths. I've been out of the loop lately, but additional "primary"
> Brillinat Horizon fluors are likely to be introduced by BD Biosciences.
>
> What is often overlooked with these fluors is that they are so bright and
> photostable that their real potential value is to use them for directly
> labelled, primary antibodies, without need for amplification and the issues
> that can arise from using secondary antibodies. I think it remains to be
> seen how useful they might ultimately be as primary probes.
>
> Perhaps the problems associated with direct primary antibody conjugation
> such as:
>
> - maintaining the antigenicity of the antibody
> - permeability into tissue due to size
>
> might be too difficult to overcome, but because of their brightness &
> photostability, it seems worth the effort to try for those folks who are
> interested in developing new/improved methodologies.
>
> Jeff
>
>
> *Jeff Carmichael*
> *Product & Technical Marketing Manager*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
> * <[hidden email]>*
>
> On Tue, Aug 21, 2018 at 8:32 AM, Adrian Smith <[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.
>> *****
>>
>> Most of the longer wavelength Brilliant Violet reagents are tandems, ie a
>> BV-polymer backbone as a donor with a second molecule as acceptor - hence
>> the excitation at 561/594/633.
>>
>> Since Sirigin was purchased by Becton Dickinson they have come out with
>> additional base polymers - Brilliant UV, Brilliant Blue, Brilliant
>> Yellow/Green (may not be commercially available yet). Generally in each
>> series the first reagent is a straight polymer and subsequent ones are
>> tandems. As I know only BD is producing these and the focus has been on
>> reagents for flow cytometry.
>>
>> http://www.bdbiosciences.com/anz/go/brilliant/
>>
>> (this is the ANZ site - US site is down for me at the moment)
>>
>> Regards,
>>
>> Adrian
>>
>>
>>
>> On Tue, 21 Aug 2018 at 22:15, Moulding, Dale <[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.
>>> *****
>>>
>>> I've tried the BV dyes. I may have got the wrong impression about them,
>>> but thought they were relatively large? Certainly for whole mount
>> staining
>>> I thought they didn’t penetrate tissue very well.
>>> I also found that while BV421 was useful, the longer wavelength BV dyes
>>> when used on monolayers gave quite considerable fluorescence when
> excited
>>> at 561, 594 or 633 nm, making them less useful than we had hoped for
>>> multichannel imaging without unmixing.
>>> I'd be very interested to hear if I'm mistaken, and they are in fact
>> small
>>> molecules that can penetrate well into whole mounts. If so we would
>>> certainly switch to BV421 instead of Alexa405. Empirically we do find
>>> alexa405 is not particularly bright, but generally find it to be as
> about
>>> as photostable as Alexa568.
>>>
>>> Cheers
>>>
>>> Dale
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List <[hidden email]> On
>>> Behalf Of Jeff Carmichael
>>> Sent: 21 August 2018 12:31
>>> To: [hidden email]
>>> Subject: Re: recommendation for a "blue" secondary fluorochrome
>> COMMERCIAL
>>> RESPONSE
>>>
>>> *****
>>> 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.
>>> *****
>>>
>>> This has been addressed more than once in the listserv, but below are
>> some
>>> resources to look into the BrilliantViolet fluorophores which are much
>> more
>>> than an order of magnitude brighter than AF405. You could potentially
> add
>>> two additional channels in this spectral "dead zone".
>>>
>>> Chroma Technology has no commercial interest in these fluorophores, only
>>> in the filter sets we've recommended for detecting them.
>>>
>>>
>>> https://www.jacksonimmuno.com/technical/products/conjugate-
>> selection/brilliant-violet
>>> http://bit.ly/ChromaTechnology2MtEdC9
>>> http://bit.ly/bdbiosciences2MEed69
>>>
>>> Good luck and please share your results!
>>> Jeff
>>>
>>> *Jeff Carmichael*
>>> *Product & Technical Marketing Manager*
>>>
>>> *[hidden email] <[hidden email]> | 802-428-2528*
>>>
>>> * <[hidden email]>*
>>>
>>> On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) <
>>> [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.
>>>> *****
>>>>
>>>> Is there a better or preferred "blue" fluorochrome? Mostly this is a
>>>> widefield fluorescence question (using a DAPI cube), but there is the
>>>> possibility for using a confocal with a 405nm laser line.
>>>>
>>>> I'm working with a group that is trying to work around a frustrating
>>>> autofluorescence issue in liver tissue. What we ended up doing on an
>>>> earlier project was not using any secondaries that fluoresced in the
>>>> green channel (no fitc/AF488/GFP) and instead used the green channel
>>>> on our widefield microscope as a control, ratioing this image so we
>>>> could subtract it out of the other channels to remove some of the
>>>> broad autofluorescent background. This worked nicely. In the published
>>>> images we showed nuclei
>>>> (blue) and two specific stains using AF568 and AF647, with reduced
>>>> background autofluorescence.
>>>>
>>>> The PI now wants to have labelling with three specific fluorochromes,
>>>> and I am advocating using the green channel as the generic "everything
>>>> lights up" channel for context, so we can hopefully skip the Hoechst
>>> 3342 or DAPI.
>>>> Since we won't be using DNA dyes, is there a good blue fluorochrome
>>>> that can be used as a secondary for specific labelling of a marker
>>>> found on an intracellular organelle (other than the nucleus)?
>>>>
>>>> Thanks,
>>>> Doug
>>>>
>>>> ------------------------------------------------------------
>>>> ------------------------------
>>>> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of
>>>> Cellular & Molecular Medicine, University of Arizona
>>>> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
>>>>
>>>> office: LSN 463 email: [hidden email]<mailto:
>>>> [hidden email]>
>>>> voice: 520-626-2824 fax: 520-626-2097
>>>>
>>>> http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
>>>> Home of: "Microscopy and Imaging Resources on the WWW"
>>>>
>>>> UA Microscopy Alliance - http://microscopy.arizona.edu<
>>>> http://microscopy.arizona.edu/>
>>>>
>>> --
>>> <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned
>>> company*
>>> 10 Imtec Lane, Bellows Falls, Vermont 05101 USA
>>> 800-824-7662 |
>>> FAX: 802-428-2525
>>> www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:
>>> [hidden email]>
>>>
>> --
>> <https://www.centenary.org.au>
>>

--

George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

Re: recommendation for a "blue" secondary fluorochrome **Vendor Reply**

Re: recommendation for a "blue" secondary fluorochrome **Vendor Reply**

Re: recommendation for a "blue" secondary fluorochrome **Vendor Reply**

Re: recommendation for a "blue" secondary fluorochrome **Vendor Reply**

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply

Re: recommendation for a "blue" secondary fluorochrome Vendor Reply