PECAM(CD31)IHC

[njcragg]

Just to update anyone who is following this...found no difference between  applying the endogenous peroxidase step before or after the Proteinase K step.

Got this antibody to work consistently as previously described (Prot K rmins RT, primary 1/50 overnight incubation), just make sure the Biotinylated Rabbit-anti-Rat is still good and on for 45 minutes.

[cranberry80]

Hello everybody
I'm also struggling to get a signal with the CD31 antibody from Pharmingen in PFA fixed sections. We fix our tissues (about 5 mm diameter) during 24h: do you think this is too much? I've seen that some labs fix small samples during only 4-8h. Could an overfixation play a role in the absence of signal?

[ImmunoNYC]

Are you using the MEC13.3 clone? If so, then YES, PFA fixation is devastating to the signal achieved with this particular clone. You will probably need to trypsinize to get some signal. Please tell us your protocol and we can help.

For future specimens ....  If you are doing frozen sections why are you fixing before sectioning? As Excalibur has said in this forum many times, it sort of defeats the whole purpose of doing a frozen section. If you NEED to fix before sectioning for some reason I suggest you fix for a shorter amount of time, say 3-4 hours at 4 deg C at MOST based on the size of your specimen.[/color]

Hello everybody
I'm also struggling to get a signal with the CD31 antibody from Pharmingen in PFA fixed sections. We fix our tissues (about 5 mm diameter) during 24h: do you think this is too much? I've seen that some labs fix small samples during only 4-8h. Could an overfixation play a role in the absence of signal?

[njcragg]

Hi,

Yes I think fixation time does affect what antigens can be retrieved.  However, we routinely fix in formal saline for 18 - 24 hours and then transfer to 70% ethanol before processing, which I thought was acutally a shorter length of time than the majority.  All the samples of mouse skin which I successfully stained with anti-mouse CD31, clone MEC13.3, were fixed in this way.   I think it is a very difficult antibody, I tried lots of different things and didn't get any staining at all, then it just began working with a relatively straightforward protocol which bemused me somewhat.

P.S. I've just realised a typo in my previous message, it should have said "Proteinase K for 5 mins at RT".

[Mariya]

Hi...just responding to a post about detecting mouse using PECAM-1 antibody.  I don't know if this will work in PEFF sections but have you considered using MECA instead?  Mouse Endothelial Cell Antigen.  Detects only mouse endothelial cells, no cross-reactivity with human.

Cheers,

Mariya

[ImmunoNYC]

MECA does work in FFPE sections and I use it routinely, it's gorgeous but it does not stain all vascular beds. CD31 is a superior marker IMO. Also b/c it is relatively unknown I have experienced resistance from reviewers . Even so, I use it all the time. It's gorgeous.[/color]

Hi...just responding to a post about detecting mouse using PECAM-1 antibody.  I don't know if this will work in PEFF sections but have you considered using MECA instead?  Mouse Endothelial Cell Antigen.  Detects only mouse endothelial cells, no cross-reactivity with human.

[ishtar]

PharMingen rat antimouse antibody worked really well for me, I used frozen sections fixed with 4%PFA.

But, I know it does NOT work on paraffin sections, our tech learnt that in a hard way...