Mouse FFPE on IMC

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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Mouse FFPE on IMC

Post by mattbliss90 » Tue Nov 12, 2019 8:06 pm

Hello All,

I am writing this as a follow-up to my post from last summer. I am wondering if anyone on this forum has had any success at establishing a mouse FFPE panel for IMC. If so what conditions do you use for the antigen retrieval, antibody incubation times etc. Our group uses the Ventana system for antigen retrieval and staining with the following setup:

- High pH 8.5 antigen retrieval for 44 minutes at 100 C
- 60 minute heated antibody incubation at 42 C

So far this has worked well for most of our clones (CD4 H129.19, CD8 4SM15, B220 RA3-6B2, IBA1 poly) using IHC after metal conjugation in mouse spleen. However once we switch to combining all the antibodies together and running on the IMC… only the B220, and IBA appear to work with a very weak signal for the CD8 (4SM15). We observe nonspecific binding for CD3 (poly, 145-2C11), PD1 (poly), CD4 (H129.19), and CD8 (53-6.7). Does anybody have any protocols, suggestions, and different clones etc. that have worked for them?

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Re: Mouse FFPE on IMC

Post by douglas.linn » Wed Nov 20, 2019 2:25 pm

Hi Matt,
We've done quite a bit of mouse IMC work with FFPE tissues. We largely abandoned frozen due to inability to get nuclear markers (Iridiums or H3) to work sufficiently while retaining good membrane staining. We have used a regular microwave or the Biogenex microwave with EDTA pH9 AR buffer (abcam). We also use the Biocare Decloaker. Most of the mouse-reactive clones we use are either from abcam, CST, or R&D. Overnight incubation has been our standard, but we haven't played much with these conditions. Most of the antibodies for our human panel work great staining 1 hr at RT, but in general the human-reactive antibodies are better than mouse in my experience. I also have found that a notable proportion of mouse-reactive antibodies do no like the conjugation chemistry which is unfortunate when we have limited options to begin with.
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Re: Mouse FFPE on IMC

Post by monirathhav » Tue Apr 28, 2020 8:12 pm

Hi Matt, can you share your B220 IMC image that you mentioned to managed to get to work on IMC?

Doug, how is your antigen retrieval and at at what temperature? Did you get B220, CD4, CD8, Keratin, MHC class I and class II to work on mouse FFPE on IMC? If you did, can you share with us the clones and tricks you use?

Thank you.

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