Immunofluorescence multiplexing with an interesting "modified hapten" labeling technology

[Jeffrey Carmichael]

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COMMERCIAL POST

In a recent conversation thread about blue fluorochromes, George McNamara
mentioned alternative labeling methods such as biotin/streptavidin and
TSA-hapten.

This reminded me of a customer we've been working with who has developed
what they're calling a "next generation" hapten (not TSA) labeling
technology with very high specificity, affinity and amplification. This in
turn means that the primary antibody species origin doesn't matter, and 4
mouse monoclonal antibodies may be used together on the same tissue sample,
including without multiple labeling steps. The "modified" haptens are
referred to as peptide bar-codes which apparently allow for significant
multiplexing:

https://cellidx.com/technology
https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892

Chroma Technology has no commercial interest in these products per se,
other than the filter sets that may be recommended for use with them.

Jeff

*Jeff Carmichael*
*Product & Technical Marketing Manager*

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

* <[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]>                             

[George McNamara]

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As much as I am a fan of TSA (and look forward to the methyl-luminol
alternative), more multiplexing options:

https://www.ncbi.nlm.nih.gov/pubmed/29263082

Pleiner et al 2018 JCB -A toolbox of anti-mouse and anti-rabbit IgG
secondary nanobodies.

https://www.ncbi.nlm.nih.gov/pubmed/29444803

Commentary

The commercial arm is www.nano-tag.com

In many ways, nanobodies are an update of the "Pretty Poly" method of
Morris and Stanley 2003
https://www.ncbi.nlm.nih.gov/pubmed/?term=pretty+poly+stanley

see also Molecular Probes Zenon product line.

These are not amplification.

See also Brilliants and SuperBrights: ideally the fluorescence
microscopy community could be using the direct labeled fluorescent
antibodies routinely used in flow cytometers and FACE sorter. I
mentioned to some visitors today that BD's X50 is named for their intent
(and look to be on target to achieve) 50plex fluorescence flow
cytometry, with several directional cell sorting (I'm spacing out on the
numbers, maybe 6 way sort now, 10 way in ~3 years?). I'm also a fan of
CyTOF, imaging CyTOF, MIBI-ToF, etcTOF.

enjoy,
George
p.s. Disclosure: I came across nano-tag.com at their exhibit at Society
for Neuroscience 2017, who turned me on to the Pleiner article. They
were nice enough to send several free samples, which our users found
worked fine on our new Leica SP8 confocal microscope (should also work
fine on our Olympus FV3000RS, but that only was installed last week).

On 8/23/2018 4:57 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.
> *****
>
> COMMERCIAL POST
>
> In a recent conversation thread about blue fluorochromes, George McNamara
> mentioned alternative labeling methods such as biotin/streptavidin and
> TSA-hapten.
>
> This reminded me of a customer we've been working with who has developed
> what they're calling a "next generation" hapten (not TSA) labeling
> technology with very high specificity, affinity and amplification. This in
> turn means that the primary antibody species origin doesn't matter, and 4
> mouse monoclonal antibodies may be used together on the same tissue sample,
> including without multiple labeling steps. The "modified" haptens are
> referred to as peptide bar-codes which apparently allow for significant
> multiplexing:
>
> https://cellidx.com/technology
> https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892
>
> Chroma Technology has no commercial interest in these products per se,
> other than the filter sets that may be recommended for use with them.
>
> Jeff
>
> *Jeff Carmichael*
> *Product & Technical Marketing Manager*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
> * <[hidden email]>*
>

--


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

[Sripad Ram-2]

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*****

Hi Jeff,
My understanding is that cellIdx technology can support upto a 4plex (i.e.
4 abs).

I dont recall seeing side by side comparison of their technology with TSA,
but what i hear is that the signal intensity is good enough to detect Ab
labeling even in notoriously autofluorescent samples (lung cancer tissue).

Sripad


On Thu, Aug 23, 2018, 2:02 PM 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.
> *****
>
> COMMERCIAL POST
>
> In a recent conversation thread about blue fluorochromes, George McNamara
> mentioned alternative labeling methods such as biotin/streptavidin and
> TSA-hapten.
>
> This reminded me of a customer we've been working with who has developed
> what they're calling a "next generation" hapten (not TSA) labeling
> technology with very high specificity, affinity and amplification. This in
> turn means that the primary antibody species origin doesn't matter, and 4
> mouse monoclonal antibodies may be used together on the same tissue sample,
> including without multiple labeling steps. The "modified" haptens are
> referred to as peptide bar-codes which apparently allow for significant
> multiplexing:
>
> https://cellidx.com/technology
> https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892
>
> Chroma Technology has no commercial interest in these products per se,
> other than the filter sets that may be recommended for use with them.
>
> Jeff
>
> *Jeff Carmichael*
> *Product & Technical Marketing Manager*
>
> *[hidden email] <[hidden email]> | 802-428-2528*
>
> * <[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]>
>

[Martin Wessendorf-2]

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*****

I'm going to take issue with George on one point: I don't think that
directly labeled primaries are necessarily the best way to go.  The
beauty of indirect immunofluorescence is that you can combine a given
primary antibody with any number of different fluorophores (in the form
of fluorophore-conjugated secondary antibodies). Once you've
characterized that primary antibody, you can be reasonably sure that it
will label the same molecule no matter what color secondary antibody
it's paired with (...assuming that the secondary works well enough to
detect any labeling!).  In contrast, each different
fluorophore-conjugate of a directly conjugated primary antibody, is a
chemically different reagent that may have different binding properties
and will need to be re-characterized for specificity.  --I.e., you would
be setting yourself up for a lot of extra work if you use directly
conjugated primaries.  (If you've already characterized the specific
reagents you'll be using, then this isn't an issue.)

My two-cents worth!

Martin Wessendorf



On 8/23/2018 8:43 PM, George McNamara 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.
> *****
>
> As much as I am a fan of TSA (and look forward to the methyl-luminol
> alternative), more multiplexing options:
>
> https://www.ncbi.nlm.nih.gov/pubmed/29263082
>
> Pleiner et al 2018 JCB -A toolbox of anti-mouse and anti-rabbit IgG
> secondary nanobodies.
>
> https://www.ncbi.nlm.nih.gov/pubmed/29444803
>
> Commentary
>
> The commercial arm is www.nano-tag.com
>
> In many ways, nanobodies are an update of the "Pretty Poly" method of
> Morris and Stanley 2003
> https://www.ncbi.nlm.nih.gov/pubmed/?term=pretty+poly+stanley
>
> see also Molecular Probes Zenon product line.
>
> These are not amplification.
>
> See also Brilliants and SuperBrights: ideally the fluorescence
> microscopy community could be using the direct labeled fluorescent
> antibodies routinely used in flow cytometers and FACE sorter. I
> mentioned to some visitors today that BD's X50 is named for their
> intent (and look to be on target to achieve) 50plex fluorescence flow
> cytometry, with several directional cell sorting (I'm spacing out on
> the numbers, maybe 6 way sort now, 10 way in ~3 years?). I'm also a
> fan of CyTOF, imaging CyTOF, MIBI-ToF, etcTOF.
>
> enjoy,
> George
> p.s. Disclosure: I came across nano-tag.com at their exhibit at
> Society for Neuroscience 2017, who turned me on to the Pleiner
> article. They were nice enough to send several free samples, which our
> users found worked fine on our new Leica SP8 confocal microscope
> (should also work fine on our Olympus FV3000RS, but that only was
> installed last week).
>
> On 8/23/2018 4:57 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.
>> *****
>>
>> COMMERCIAL POST
>>
>> In a recent conversation thread about blue fluorochromes, George
>> McNamara
>> mentioned alternative labeling methods such as biotin/streptavidin and
>> TSA-hapten.
>>
>> This reminded me of a customer we've been working with who has developed
>> what they're calling a "next generation" hapten (not TSA) labeling
>> technology with very high specificity, affinity and amplification.
>> This in
>> turn means that the primary antibody species origin doesn't matter,
>> and 4
>> mouse monoclonal antibodies may be used together on the same tissue
>> sample,
>> including without multiple labeling steps. The "modified" haptens are
>> referred to as peptide bar-codes which apparently allow for significant
>> multiplexing:
>>
>> https://cellidx.com/technology
>> https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892
>>
>> Chroma Technology has no commercial interest in these products per se,
>> other than the filter sets that may be recommended for use with them.
>>
>> Jeff
>>
>> *Jeff Carmichael*
>> *Product & Technical Marketing Manager*
>>
>> *[hidden email] <[hidden email]> | 802-428-2528*
>>
>> * <[hidden email]>*
>>
>

[George McNamara]

*****
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*****

Hi Martin,

I chickened out on including (Komodo) dragon IgG in the (thought
experiment, not 'reduced to practice') table from our immunofluorescence
microscopy chapter (2011, book 1 chp 15 of
https://www.cshlpress.com/default.tpl?action=full&--eqskudatarq=872&typ=ps&newtitle=Imaging%3A%20A%20Laboratory%20Manual 
... recycled from an earlier imaging book)

Tubulin               Mouse IgG1     Goat–anti-mouse IgG1 Alexa Fluor
488 Green
Actin                   Guinea pig IgG Goat–anti-guinea pig-IgG Lucifer
Yellow Yellow
Vimentin            Rat mAb IgM  Goat–anti-rat-IgM Cy3B Orange
Cytokeratin        Mouse IgG2a  Goat–anti-mouse IgG2a DyLight 594 (or
Texas Red) Red
Neurofilament  Chicken IgY     Goat–anti-chicken IgY HiLyte Fluor 647
(or Cy5) Far red
Nestin                 Rabbit IgG      Goat–anti-rabbit IgG Atto 680
Infrared
CD31                   Donkey IgG    Goat–anti-donkey IgG IRDye800 Infrared

Quite unwieldy!

for comparison, clinical flow cytometry is now 'routinely' done with a
lot more than 7plex direct labeled antibodies.

As for tissue section autofluorescence vs direct labeled antibody
brightness: there are now many ways to decrease autofluorescence before
imaging (I seem to be getting ads from Biotium once a week about their
stuff, for example ... various authors have published using UV
transilluminator to good and/or poor effect). There is also potential
for new (likely expensive) fluorescence microscopes, such as Leica SP8
FALCON (disclosure: our image core hosts FALCON workshop week of Sept
10, 2018 ... hmmm: maybe I should measure BV421 lifetime), or other Fast
FLIM instruments (whether upgrades or new). For examples, if NADH,
collagen, and elastin autofluorescence lifetimes are 1 nanosecond, and
organic dyes of similar spectra are each ~4 ns, could be 6plex just in
blues and greens, plenty of spectra for more.

enjoy,
George


On 8/23/2018 10:33 PM, Martin Wessendorf 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'm going to take issue with George on one point: I don't think that
> directly labeled primaries are necessarily the best way to go.  The
> beauty of indirect immunofluorescence is that you can combine a given
> primary antibody with any number of different fluorophores (in the
> form of fluorophore-conjugated secondary antibodies). Once you've
> characterized that primary antibody, you can be reasonably sure that
> it will label the same molecule no matter what color secondary
> antibody it's paired with (...assuming that the secondary works well
> enough to detect any labeling!).  In contrast, each different
> fluorophore-conjugate of a directly conjugated primary antibody, is a
> chemically different reagent that may have different binding
> properties and will need to be re-characterized for specificity. 
> --I.e., you would be setting yourself up for a lot of extra work if
> you use directly conjugated primaries.  (If you've already
> characterized the specific reagents you'll be using, then this isn't
> an issue.)
>
> My two-cents worth!
>
> Martin Wessendorf
>
>
>
> On 8/23/2018 8:43 PM, George McNamara 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.
>> *****
>>
>> As much as I am a fan of TSA (and look forward to the methyl-luminol
>> alternative), more multiplexing options:
>>
>> https://www.ncbi.nlm.nih.gov/pubmed/29263082
>>
>> Pleiner et al 2018 JCB -A toolbox of anti-mouse and anti-rabbit IgG
>> secondary nanobodies.
>>
>> https://www.ncbi.nlm.nih.gov/pubmed/29444803
>>
>> Commentary
>>
>> The commercial arm is www.nano-tag.com
>>
>> In many ways, nanobodies are an update of the "Pretty Poly" method of
>> Morris and Stanley 2003
>> https://www.ncbi.nlm.nih.gov/pubmed/?term=pretty+poly+stanley
>>
>> see also Molecular Probes Zenon product line.
>>
>> These are not amplification.
>>
>> See also Brilliants and SuperBrights: ideally the fluorescence
>> microscopy community could be using the direct labeled fluorescent
>> antibodies routinely used in flow cytometers and FACE sorter. I
>> mentioned to some visitors today that BD's X50 is named for their
>> intent (and look to be on target to achieve) 50plex fluorescence flow
>> cytometry, with several directional cell sorting (I'm spacing out on
>> the numbers, maybe 6 way sort now, 10 way in ~3 years?). I'm also a
>> fan of CyTOF, imaging CyTOF, MIBI-ToF, etcTOF.
>>
>> enjoy,
>> George
>> p.s. Disclosure: I came across nano-tag.com at their exhibit at
>> Society for Neuroscience 2017, who turned me on to the Pleiner
>> article. They were nice enough to send several free samples, which
>> our users found worked fine on our new Leica SP8 confocal microscope
>> (should also work fine on our Olympus FV3000RS, but that only was
>> installed last week).
>>
>> On 8/23/2018 4:57 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.
>>> *****
>>>
>>> COMMERCIAL POST
>>>
>>> In a recent conversation thread about blue fluorochromes, George
>>> McNamara
>>> mentioned alternative labeling methods such as biotin/streptavidin and
>>> TSA-hapten.
>>>
>>> This reminded me of a customer we've been working with who has
>>> developed
>>> what they're calling a "next generation" hapten (not TSA) labeling
>>> technology with very high specificity, affinity and amplification.
>>> This in
>>> turn means that the primary antibody species origin doesn't matter,
>>> and 4
>>> mouse monoclonal antibodies may be used together on the same tissue
>>> sample,
>>> including without multiple labeling steps. The "modified" haptens are
>>> referred to as peptide bar-codes which apparently allow for significant
>>> multiplexing:
>>>
>>> https://cellidx.com/technology
>>> https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892 
>>>
>>>
>>> Chroma Technology has no commercial interest in these products per se,
>>> other than the filter sets that may be recommended for use with them.
>>>
>>> Jeff
>>>
>>> *Jeff Carmichael*
>>> *Product & Technical Marketing Manager*
>>>
>>> *[hidden email] <[hidden email]> | 802-428-2528*
>>>
>>> * <[hidden email]>*
>>>
>>

--


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

[Jeffrey Carmichael]

*****
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*****

Hi Sripad,

I don't recall seeing a direct comparison of image data with TSA-prepared
samples either, but I'd hesitate to comment on that given that I haven't
performed this processing myself.

The TSA protocols I'm familiar with involve sequential labeling steps
involving stripping by microwaving tissue samples between each successive
round, and overall involve a very large number of total processing steps. I
can't help but be skeptical of these protocols. I know there are multiple
TSA protocols, but I'm not very familiar with them.

Part of what intuitively appeals to me about this peptide-hapten approach
is it's simplicity and relative ease, which I would expect to aid
reproducibility, especially when also considering the high specificity of
the secondary reagents.

I have seen data that I'm not at liberty to disclose, and I also lack very
detailed knowledge about the technology to discuss intelligently, so I'd
defer to David Schwartz at CellIDx to respond.

Jeff



On Thu, Aug 23, 2018 at 9:44 PM, S Ram <[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,
> My understanding is that cellIdx technology can support upto a 4plex (i.e.
> 4 abs).
>
> I dont recall seeing side by side comparison of their technology with TSA,
> but what i hear is that the signal intensity is good enough to detect Ab
> labeling even in notoriously autofluorescent samples (lung cancer tissue).
>
> Sripad
>
>
> On Thu, Aug 23, 2018, 2:02 PM 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.
> > *****
> >
> > COMMERCIAL POST
> >
> > In a recent conversation thread about blue fluorochromes, George McNamara
> > mentioned alternative labeling methods such as biotin/streptavidin and
> > TSA-hapten.
> >
> > This reminded me of a customer we've been working with who has developed
> > what they're calling a "next generation" hapten (not TSA) labeling
> > technology with very high specificity, affinity and amplification. This
> in
> > turn means that the primary antibody species origin doesn't matter, and 4
> > mouse monoclonal antibodies may be used together on the same tissue
> sample,
> > including without multiple labeling steps. The "modified" haptens are
> > referred to as peptide bar-codes which apparently allow for significant
> > multiplexing:
> >
> > https://cellidx.com/technology
> > https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892
> >
> > Chroma Technology has no commercial interest in these products per se,
> > other than the filter sets that may be recommended for use with them.
> >
> > Jeff
> >
> > *Jeff Carmichael*
> > *Product & Technical Marketing Manager*
> >
> > *[hidden email] <[hidden email]> | 802-428-2528*
> >
> > * <[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]>
> >
>

--
 <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]>                             

[Jim Mansfield]

*****
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*****

Hi,

I have heard very good things about Cell IDx's multiplexing methodology, so I am glad to hear that word of it is spreading. I'd love to hear if anyone has any hands-on experience comparing it to TSA (either PerkinElmer's Opal or Roche's TSA variant).

But since this thread is partly about multiplex IHC methods, I thought I'd mention Ultivue, who has developed their own methodology for doing multiplex IHC staining:

https://www.ultivue.com/

I have not tried either Cell IDx or Ultivue, so I can't give any comments on how they compare - but I'd love to hear from someone who has!

Best,

Jim
Andor Technology

Commercial interest: I have no commercial interest in any of these methods ... although in the spirit of full disclosure, I used to work at PerkinElmer in their Quantitative Pathology group and am very familiar with the TSA methods.


Jim Mansfield
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeff Carmichael
Sent: Thursday, August 23, 2018 3:57 PM
To: [hidden email]
Subject: Immunofluorescence multiplexing with an interesting "modified hapten" labeling technology

*****
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*****

COMMERCIAL POST

In a recent conversation thread about blue fluorochromes, George McNamara
mentioned alternative labeling methods such as biotin/streptavidin and
TSA-hapten.

This reminded me of a customer we've been working with who has developed
what they're calling a "next generation" hapten (not TSA) labeling
technology with very high specificity, affinity and amplification. This in
turn means that the primary antibody species origin doesn't matter, and 4
mouse monoclonal antibodies may be used together on the same tissue sample,
including without multiple labeling steps. The "modified" haptens are
referred to as peptide bar-codes which apparently allow for significant
multiplexing:

https://cellidx.com/technology
https://event.webcasts.com/viewer/event.jsp?ei=1187871&tp_key=9db65f5892

Chroma Technology has no commercial interest in these products per se,
other than the filter sets that may be recommended for use with them.

Jeff

*Jeff Carmichael*
*Product & Technical Marketing Manager*

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

* <[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]>                            

[Sripad Ram-2]

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Hi Jim,
Glad to note that you are mentioning Ultivue. On the same spirit, we should
also mention Akoya biosciences which is a spinoff of Gary Nolan's CODEX
technology. Both use dna labeled primary Abs with slight variation in
washing strategies. As for a direct comparison between Ultivue and Perkin
Elmer, that is something that we are potentially interested and may pursue
at some point in time.

Jeff,
I am very familiar with perkin elmer Opal dyes and have been using them for
well over 2 years. There are ways you can do Ab removal without doing
microwave treatment. However you'd need an auto stainer for this (such as a
leica bond rx or similar device).
Opal dyes have their challenges but its not difficult to have the right
controls to check for tissue loss/damage. The bigger challenges are with
spectral unmixing and the downstream image analysis pipelines.

Sripad

[Jeffrey Carmichael]

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Sripad and Jim,

Yes, the DNA barcoding approach is potentially very powerful. I'm not very
familiar with it but I see this paper, published this month in Cell:
https://www.cell.com/cell/pdf/S0092-8674(18)30904-8.pdf

Otherwise, my experience is that simplicity will aid reproducibility.
That's partly what is attractive to me about the modified-hapten approach.
It sounds like you have more patience than I do regarding complex sample
processing!

I'm curious what the utility of these various techniques will be in
applications ranging from basic research to preclinical to highly
multiplexed clinical data acquisition. All have somewhat different aims &
requirements.

Jeff



On Sat, Aug 25, 2018 at 1:57 AM, S Ram <[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 Jim,
> Glad to note that you are mentioning Ultivue. On the same spirit, we should
> also mention Akoya biosciences which is a spinoff of Gary Nolan's CODEX
> technology. Both use dna labeled primary Abs with slight variation in
> washing strategies. As for a direct comparison between Ultivue and Perkin
> Elmer, that is something that we are potentially interested and may pursue
> at some point in time.
>
> Jeff,
> I am very familiar with perkin elmer Opal dyes and have been using them for
> well over 2 years. There are ways you can do Ab removal without doing
> microwave treatment. However you'd need an auto stainer for this (such as a
> leica bond rx or similar device).
> Opal dyes have their challenges but its not difficult to have the right
> controls to check for tissue loss/damage. The bigger challenges are with
> spectral unmixing and the downstream image analysis pipelines.
>
> Sripad
>

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