60plex immunofluorescence microscopy

[George McNamara]

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Dear Confocal listserv members,

No need to have plex envy when your flow cytometry colleagues talk
about  ~20plex in fluorescence flow cytometry or ~40plex in CyTOF mass
cytometry (or 10plex on the EMD Millipore/AMNIS ImageStreamX imaging in
flow cytometer). Here are the links to recent papers on "60plex"
immunofluorescence microscopy (and should be able to do more).


new Gerdes et al GE  multiplex paper in PNAS (open access)

http://www.pnas.org/content/early/2013/06/27/1300136110.full.pdf

Gerdes PNAS supplemental files are at
http://www.pnas.org/content/early/2013/06/27/1300136110/suppl/DCSupplemental 


And another Gerdes (Nelson) et al multiplex paper (also open access)
http://bio.biologists.org/content/2/5/439.abstract

In addition to GE, see
http://www.chipcytometry.com/technology.phtml


Enjoy,

George

[Kilgore, Jason-2]

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Interesting.  61 different targets on the same paraffin section.  

To be somewhat pedantic, when I think of "plex"-ing I think of concurrent labeling.  Really, as I understand it, this example is sequential labeling and imaging, with a patented "fluorophore inactivation" solution in between each labeling and imaging cycle, with a cy dye.  You still have to deal with autofluorescence and taking the exact same view for each imaging session to overlay.

But a very novel method.

Jason


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Tuesday, July 02, 2013 6:14 PM
To: [hidden email]
Subject: 60plex immunofluorescence microscopy

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear Confocal listserv members,

No need to have plex envy when your flow cytometry colleagues talk
about  ~20plex in fluorescence flow cytometry or ~40plex in CyTOF mass
cytometry (or 10plex on the EMD Millipore/AMNIS ImageStreamX imaging in
flow cytometer). Here are the links to recent papers on "60plex"
immunofluorescence microscopy (and should be able to do more).


new Gerdes et al GE  multiplex paper in PNAS (open access)

http://www.pnas.org/content/early/2013/06/27/1300136110.full.pdf

Gerdes PNAS supplemental files are at
http://www.pnas.org/content/early/2013/06/27/1300136110/suppl/DCSupplemental 


And another Gerdes (Nelson) et al multiplex paper (also open access)
http://bio.biologists.org/content/2/5/439.abstract

In addition to GE, see
http://www.chipcytometry.com/technology.phtml


Enjoy,

George

[JOEL B. SHEFFIELD]

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To which I would add that each of the antibodies must be labeled directly
--no secondary ab. Nevertheless, quite a tour de force.

Joel


On Wed, Jul 3, 2013 at 12:18 PM, Kilgore, Jason
<[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Interesting.  61 different targets on the same paraffin section.
>
> To be somewhat pedantic, when I think of "plex"-ing I think of concurrent
> labeling.  Really, as I understand it, this example is sequential labeling
> and imaging, with a patented "fluorophore inactivation" solution in between
> each labeling and imaging cycle, with a cy dye.  You still have to deal
> with autofluorescence and taking the exact same view for each imaging
> session to overlay.
>
> But a very novel method.
>
> Jason
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of George McNamara
> Sent: Tuesday, July 02, 2013 6:14 PM
> To: [hidden email]
> Subject: 60plex immunofluorescence microscopy
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Confocal listserv members,
>
> No need to have plex envy when your flow cytometry colleagues talk
> about  ~20plex in fluorescence flow cytometry or ~40plex in CyTOF mass
> cytometry (or 10plex on the EMD Millipore/AMNIS ImageStreamX imaging in
> flow cytometer). Here are the links to recent papers on "60plex"
> immunofluorescence microscopy (and should be able to do more).
>
>
> new Gerdes et al GE  multiplex paper in PNAS (open access)
>
> http://www.pnas.org/content/early/2013/06/27/1300136110.full.pdf
>
> Gerdes PNAS supplemental files are at
>
> http://www.pnas.org/content/early/2013/06/27/1300136110/suppl/DCSupplemental
>
>
> And another Gerdes (Nelson) et al multiplex paper (also open access)
> http://bio.biologists.org/content/2/5/439.abstract
>
> In addition to GE, see
> http://www.chipcytometry.com/technology.phtml
>
>
> Enjoy,
>
> George
>

--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs

[George McNamara]

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Hi Joel and Jason,

GE is the manufacturer of the CyDyes (though the patents should be up by
now, so could become generic). A lot of direct labeled antibodies -
though I wonder if the many "blank" channels were bad antibodies that
were simply not mentioned in the paper. The flow cytometry field mostly
uses direct labeled antibodies, so this experiment could have been done
with purchased reagents.

Their "alkaline H2O2" should also work on Cy5.5, Cy7 (filter cube
mentioned on one of the Gerdes et al microscopes) and Alexa Fluor 647
(just different enough chemical structure from Cy5 that hopefully
prevented patent lawyers from making too much money ... instead Jason's
packaging and shipping depts are the profit center for AF647).

GE's patent (my thanks to Badri Roysam, UHouston, for digging out the
relevant passage) is below.

    http://www.google.com/patents/US7741046

    Example 9 Dye Destruction, Staining, and Imaging

    NaOH solution and H_2 O_2 solution were used for signal destruction.
    A NaOH solution was prepared using 500 ?L of 50 volume percent NaOH
    and 49.5 mL of PBS. The final pH of the NaOH solution was around
    11.9-12.5. A H_2 O_2 solution was prepared by mixing 10 mL of 0.5M
    sodium carbonate (pH 10), 5 mL of 30 volume percent H_2 O_2 , and 35
    mL of water. A slide was placed in the NaOH or H_2 O_2 solution for
    15 minutes with gentle agitation. After 15 minutes, the slide was
    washed again with PBS, cover slipped and either imaged again
    (optional) to check the efficacy of the dye destruction or restained
    and imaged. Restaining and reimaging steps were carried out using
    the process described in Example 8. Following imaging, a slide was
    subjected to signal destruction, staining, and imaging cycles, and
    the process was repeated a multiple number of times. The tissue
    samples were imaged using 1-9 different antibodies. After imaging
    with the cyanine series, the slide was optionally stained and imaged
    with morphological stains H&E.

The above reads rather strange in that the first sentence states (my
capitals) "NaOH solution AND H_2 O_2 solution", then later, "slide was
placed in the NaOH OR H_2 O_2 solution", which sounds like either NaOH
(pH ~12) OR H2O2 (pH 10) were used. Destroying dyes with high alkalinity
has probably been around since the Mauve.

Among other methods ... Gerner ... Germain 2012 Immunity did a few plex
on a standard confocal. Dr. Dragan Maric, NIH, uses 10 fluorophores per
cycle, has done at least 3 cycles. Gerdes et al cite the 7plex paper
from Tsurui et al (done with a SKY system, though 3 acquisitions of 2+2+3).


On 7/3/2013 11:39 AM, JOEL B. SHEFFIELD wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> To which I would add that each of the antibodies must be labeled directly
> --no secondary ab. Nevertheless, quite a tour de force.
>
> Joel
>
>
> On Wed, Jul 3, 2013 at 12:18 PM, Kilgore, Jason
> <[hidden email]>wrote:
>
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Interesting.  61 different targets on the same paraffin section.
>>
>> To be somewhat pedantic, when I think of "plex"-ing I think of concurrent
>> labeling.  Really, as I understand it, this example is sequential labeling
>> and imaging, with a patented "fluorophore inactivation" solution in between
>> each labeling and imaging cycle, with a cy dye.  You still have to deal
>> with autofluorescence and taking the exact same view for each imaging
>> session to overlay.
>>
>> But a very novel method.
>>
>> Jason
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of George McNamara
>> Sent: Tuesday, July 02, 2013 6:14 PM
>> To: [hidden email]
>> Subject: 60plex immunofluorescence microscopy
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Confocal listserv members,
>>
>> No need to have plex envy when your flow cytometry colleagues talk
>> about  ~20plex in fluorescence flow cytometry or ~40plex in CyTOF mass
>> cytometry (or 10plex on the EMD Millipore/AMNIS ImageStreamX imaging in
>> flow cytometer). Here are the links to recent papers on "60plex"
>> immunofluorescence microscopy (and should be able to do more).
>>
>>
>> new Gerdes et al GE  multiplex paper in PNAS (open access)
>>
>> http://www.pnas.org/content/early/2013/06/27/1300136110.full.pdf
>>
>> Gerdes PNAS supplemental files are at
>>
>> http://www.pnas.org/content/early/2013/06/27/1300136110/suppl/DCSupplemental
>>
>>
>> And another Gerdes (Nelson) et al multiplex paper (also open access)
>> http://bio.biologists.org/content/2/5/439.abstract
>>
>> In addition to GE, see
>> http://www.chipcytometry.com/technology.phtml
>>
>>
>> Enjoy,
>>
>> George
>>
>>      
>
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054