IMC staining - blocking

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monirathhav
Posts: 11
Joined: Sat Feb 24, 2018 6:33 am

IMC staining - blocking

Post by monirathhav » Thu Apr 26, 2018 6:16 pm

Hi,

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

Thank you.

Monirath
RaulCatena
Posts: 25
Joined: Mon Nov 27, 2017 12:49 pm

Re: IMC staining - blocking

Post by RaulCatena » Thu Apr 26, 2018 6:44 pm

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
ssivajothi
Posts: 30
Joined: Thu Jan 11, 2018 9:00 pm

Re: IMC staining - blocking

Post by ssivajothi » Thu Apr 26, 2018 10:28 pm

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
RaulCatena
Posts: 25
Joined: Mon Nov 27, 2017 12:49 pm

Re: IMC staining - blocking

Post by RaulCatena » Fri Apr 27, 2018 10:49 am

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
douglas.linn
Posts: 11
Joined: Mon Jan 22, 2018 4:20 pm

Re: IMC staining - blocking

Post by douglas.linn » Fri May 25, 2018 6:06 pm

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.
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