PECAM(CD31)IHC

[weneng]

Happy new year to you all!
Currently I am asked to do PECAM IHC on FFPE mouse tissue. I tried Santa Cruz antibody sc-1506, Lot #L0904, using SABC method and DAKO Envision system after antigen retrieval. But none of them worked. Then PharMingen rat antimouse antibody was recommended to me. The batch numbers are 01416 and MO70717. My question is has anyone here tried these antibodies yet? Does it work? Or have you ever tried any other good PECAM antibody,CD31?

Thanks in advance!
Wen

[richard03]

The PharMingen rat anti-mouse antibody works well but ONLY on Zinc-fixed tissue sections or acetone fixed, frozen sections on my hand.

However, there was a published paper using PharMingen's CD31 antibody for FFPE sections and it worked well (according to the paper). It requires proteinase K digestion pretreatment and higher temperature incubation (37 C) for both primary and secondary antibody.

If I can find the paper, I will post it here.

Richard.

[ImmunoNYC]

Richard, were you able to find this paper? I would love to try the protocol they used to see if it works in my hands. Thanks.[/color]

[richard03]

MaximinaNYC,

Here is the full text paper I was refering to. I lost the detailed protocol that Dr. Wendy J. Huss sent to my friend and forwarded to me some years ago. But I still remember the key point is that she was using 37 C indubation for both primary and secondary antibody.

Secondly, she was using 4% paraformaldehyde (not formalin). As I have tried formalin fixed mouse tissue and it did not work. Is 4% paraformaldehyde really makes huge difference comparing with formalin? I have no clue.

Anyway here is the paper and you may contact the auther (Dr. Huss) directly and she may send your the detailed protocol.

http://cancerres.aacrjournals.org/cgi/content/full/61/6/2736

Richard

[ImmunoNYC]

Thanks Richard, I will look into it. You know I really believed there are no differences between 4% PFA and 10% NBF formalin as chemically I believed them to be the same thing. However practically I think there are differences, but I just don't know what.

[Mariya]

Hi, I've been lurking here for a while.  I tried the PECAM staining using Prot K retrival as y'all suggested.  It was great.  I had tried several other methods first and none of them worked at all.  

Here is a very summerized version of what I did.  Assume the usual blocking & washing steps etc:

Pulled the reference you suggested (great)

Mouse tissue, formalin fixed and paraffin embedded.

antigen retrieval (see IHCworld Protease K) for 15 min at 37C, then rest at RT for 10 min.

1 mAb- BD Pharmingen rat anti-mouse IgG2a (clone 13.3) - 1:50 dil (also did 1:25 but did not see a difference)

O/N @ 4C then 30 min @ 37C

2ndary - biotinylated rabbit anti-rat -1:400 dil, 1 hr @ RT

Vectastain, DAB.

I can send you a pic if need be.

Thanks for the advice, I hope this helps!

-M

[richard03]

Hi Mariya,

Very glad to hear your success in staining CD31 using formalin fixed mouse tissue. It would be wonderful if you could share your images with us. Thanks.

Do you think which step is the key? Proteinase K digestion or overnight + 37 C incubation for primary antibody?

Richard

[ImmunoNYC]

This is EXCITING news! What CONCENTRATION (not what DILUTION) of primary did you use? And precisely which catalog # was it? THANKS!!!

1 mAb- BD Pharmingen rat anti-mouse IgG2a (clone 13.3) - 1:50 dil (also did 1:25 but did not see a difference)

[Mariya]

Hi again,

Sorry I didn't think to check this forum for a bit.  So the catalog number for the antibody that worked for me is: 550274 and they have the concentration listed as 15.6 ug/mL.  I used 0.3 and 0.6 ug/mL and they both worked.

How do I send a picture?

-M

[Mariya]

Sorry Richard, I forgot to reply to your question.

Really, I don't know if either step is dispensable.  I do know that I tried PECAM with another antigen retrieval method (pH and heat) and even with the O/N incubation it didn't work.

Happily, the sections have no background which can sometimes result from antigen retrieval.

Normally I try optimizing my protocols in a more organized fashion.  First an atibody dilution test and then moving to antigen retrieval if it doesn't work or more stringent conditions if the background is high.  I have to be honest, I was so sick of getting back crystal clear, blank, slides (I mean NO background even!) from my PECAM staining that I tried everything at once.

Finally, a word about fixation.  We used 10% formaldehyde, the no methanol immunocytochemistry grade from TEM.  O/N then switched to 70% EtOH.  The sections are from granuloma tissue in and around surgical sponges which have been implanted into wt mice for two weeks so they have a lot of new vasculature.

-M

[richard03]

Go to image gallery http://www.ihcworld.com/imagegallery/

Register if you haven't, login, click upload, then just follow instruction.
Thanks.

How do I send a picture?

-M

[Mariya]

Well, I registered on the image part of the site (why do you have to register twice?) and it won't let me log in.  It won't give me a new password because it says that I don't exist as a user BUT when I try to register again it says that I can't because somebody already has my e-mail address (me...it's me, I have MY e-mail address).  And somebody already has my Login  (me....I have MY log in).  But  "it" still tells me "selected user does not exist!"  when I put in either the e-mail or the log in.

-M

[richard03]

You have to check your email and click the link sent to your email to activate your membership. Then you will be able to login and upload images.

Richard

Well, I registered on the image part of the site (why do you have to register twice?) and it won't let me log in.  It won't give me a new password because it says that I don't exist as a user BUT when I try to register again it says that I can't because somebody already has my e-mail address (me...it's me, I have MY e-mail address).  And somebody already has my Login  (me....I have MY log in).  But  "it" still tells me "selected user does not exist!"  when I put in either the e-mail or the log in.

-M

[Linesider]

The santa-cruz antibody works on routine 10% NBF sections,  if you increase the antigen retrieval time to 60 minutes(yes 60 minutes) in pressure cooker.  It was the only way I found to get reproducible staining of vessels.
I have not tried the pro-K, but some of my associates have with the 13.3 pecam clone and have had sucess.
cheers

[njcragg]

Hi,  does anybody know whether the CD31, rat monoclonal (clone MEC13.3) is specific only to mouse?  

Need to label mouse endothelial cells in human skin xenografts, separately from human endothelial cells.  

Or is there a way of blocking the human during labelling for mouse?

Thanks,

Nicola