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IMC staining - blocking

Posted: Thu Apr 26, 2018 6:16 pm
by monirathhav
Hi,

Can anyone share which blocking solutions work well for IMC staining?

Thank you.

Monirath

Re: IMC staining - blocking

Posted: Thu Apr 26, 2018 6:44 pm
by RaulCatena
Hi Monirath

I recommend Serum (FBS, Horse...) 0.5-1% and FcBlock (normally 1:100-1:500 if you use Miltenyi’s). The latter one is specially important if you go after immune cells.

Regards,

Raúl

Re: IMC staining - blocking

Posted: Thu Apr 26, 2018 10:28 pm
by ssivajothi
Hi Raul,

Do you block with serum and Fc block in successive steps or mix both in one step?

Will any flow cytometry Fc block (like Biolegend trustain fx) work or do you prefer a particular one for tissues?

Thanks,
Santhosh

Re: IMC staining - blocking

Posted: Fri Apr 27, 2018 10:49 am
by RaulCatena
Hi Santhosh,

Theory says that it would be better to do FcBlock first, and then Serum separate. Reason is that mixing them could lead to inactivation of the Fc's that block CD16/32 etc. However, I tried once and did not see anything different, but to be honest with you, I did not do a very through comparison, and I get good results anyway by adding them together. Perhaps if you are willing to try and let us know your experience... :)

I have used traditionally Miltenyi for no reason at all, just inertia from my old lab. Any brand should work just as fine, but have no idea about the right dilutions for them.

Regards,

Raúl

Re: IMC staining - blocking

Posted: Fri May 25, 2018 6:06 pm
by douglas.linn
I would be very surprised if Fc blockade was necessary for IMC as it is not in most standard IHC assay protocols. In fact, even though the serum (or BSA) blocking is traditionally thought to block FcR during IHC/IF/IMC, an interesting paper found no nonspecific staining when omitting the blocking step altogether: https://www.nature.com/articles/srep00028. In FACS or suspension CyTOF when staining is typically occurs prior to fixation, it is a different story regarding Fc blocking.