IMC on FFPE kidney

Does this antibody work? Designing a new panel?
Antibodies, isotopes, etc. for Imaging Mass Cytometry applications
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MariekeIJsselsteijn
Posts: 10
Joined: Mon Jan 15, 2018 10:52 am

IMC on FFPE kidney

Post by MariekeIJsselsteijn » Mon Jan 06, 2020 12:58 pm

Dear all,

we recently published our 40 marker IMC panel for FFPE tissue and have been using it for the characterisation of colon, lung, breast, head and neck cancer etc. and it worked well on all tissues.
However, when applying the panel on kidney tissue the majority of markers give a very poor signal. Does anyone have experience with IMC on kidney and do you perform any different steps in the staining procedure specific for kidney tissue?

Thanks alot,

Marieke

also for anyone interested, below the reference to our paper:

Ijsselsteijn ME, van der Breggen R, Farina Sarasqueta A, Koning F and de Miranda NFCC (2019) A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry. Front. Immunol. 10:2534. doi: 10.3389/fimmu.2019.02534
douglas.linn
Posts: 18
Joined: Mon Jan 22, 2018 4:20 pm

Re: IMC on FFPE kidney

Post by douglas.linn » Mon Jan 06, 2020 5:10 pm

Hi Marieke,
First of all, I really enjoyed your paper, excellent work! We have extensively validated a lot of human antibodies using normal tissue TMAs and did not observe anything unusual specific to kidney compared to any other normal tissues. With commercially available TMAs, I've found the spot-to-spot quality can be drastically different, and H&E of an adjacent section doesn't always help predict tissue quality for IMC. My best guess is tissue quality is the issue here, have you done any other analyses (ex IF) to QC the tissues?
-Doug
ssivajothi
Posts: 33
Joined: Thu Jan 11, 2018 9:00 pm

Re: IMC on FFPE kidney

Post by ssivajothi » Mon Jan 06, 2020 7:16 pm

Hi Marieke,

Echoing Doug's point, I haven't seen anything different about kidney tissue that should cause a problem. We have a single cell transcriptomics paper on human kidney up on bioRxiv, which unfortunately was put on the back burner so not published yet. I validated a number of antibodies on kidney tissue for this project and made some limited IMC panels (~12-15 markers). The staining quality was fine with the usual procedures. There might be something with the tissue used as Doug suggested.

Santhosh
MariekeIJsselsteijn
Posts: 10
Joined: Mon Jan 15, 2018 10:52 am

Re: IMC on FFPE kidney

Post by MariekeIJsselsteijn » Mon Jan 13, 2020 10:19 am

Hi Doug and santhosh,

Thanks alot for sharing your point of view. we now indeed asked our collaborators to give some more of their slides so we can do some regular IHCs to confirm that this tissue is the problem.
As soon as we have done that i'll share the results with the forum.

Marieke
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