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Mouse FFPE antibodies

Posted: Fri Aug 17, 2018 6:26 pm
by mattbliss90
Hello,
I am trying to establish a basic antibody panel to be used in mouse FFPE spleen but have seen very poor results. Has anybody had any success conjugating their own antibodies from established clones in-house and using them on mouse FFPE sections? So far some of the antibody clones we have tried include CD4 (H129.19), CD19 (6D5), CD8 (4SM15), F4/80 (Cl:A3) all produced poor results with minimal signal from the CD8 antibody. Our group has had good success conjugating CyTOF antibody clones and using them in frozen mouse tissues so I don't believe its a problem our max- par conjugation procedures. We have also run human FFPE samples using antibodies bought from Fluidigm and had good success as well.

Thanks,
Matt

Re: Mouse FFPE antibodies

Posted: Mon Aug 20, 2018 11:57 am
by hartlandj
Hi Matt,
Are these clones established for FFPE imaging? Signal strength can be an issue as there is no enzymatic signal amplification, but I haven't had any major problems conjugating antibodies for mouse FFPE tissues. I have not tried any of those clones, but because of the different antigen retrieval the clones that work in suspension or on frozen tissues are not the same clones that work with FFPE tissues.
Cheers,
Hart

Re: Mouse FFPE antibodies

Posted: Mon Aug 20, 2018 1:50 pm
by ssivajothi
Hi Matt,

Adding to Hart's response, the issue is not with the conjugation procedure but the specific clones you are using. Antigens on FFPE tissues are slightly altered and flow antibodies which usually work on frozen sections will not work on FFPE tissue. You need to source and test clones known to specifically detect these antigens on FFPE tissue.

Try the following clones:
CD19- 6OMP31
CD4- 4SM95
CD8- C8/144B
F4/80- BM8

Let me know how this works out or if you have other questions.

Best,
Santhosh

Re: Mouse FFPE antibodies

Posted: Mon Aug 20, 2018 11:36 pm
by mattbliss90
Hart and Santhosh,
The clones I listed above have been previously validated and are routinely used in the histology core facility at my company for IHC on mouse FFPE sections.

Matt

Re: Mouse FFPE antibodies

Posted: Tue Sep 18, 2018 5:26 pm
by douglas.linn
Matt,
In my hands CD19 (6D5) doesn't work for FFPE mouse tissue, but does for frozen. CD8 (4SM15) works fine for me though. The other clones you mentioned I haven't tested but overall I tend to prefer abcam & CST clones for mousef-reactive FFPE antibodies.
-Doug

Re: Mouse FFPE antibodies

Posted: Fri Oct 26, 2018 4:05 pm
by ssivajothi
Matt,

Just wanted to ask if you were able to troubleshoot your issue with the mouse FFPE antibodies working?

Santhosh