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Author Topic: PECAM(CD31)IHC  (Read 43747 times)

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Offline Linesider

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PECAM(CD31)IHC
« Reply #15 on: February 06, 2006, 11:02:10 AM »
In our hands we did not see reactivity of the 13.3 clone to human endothelial cells.
bw

PECAM(CD31)IHC
« Reply #15 on: February 06, 2006, 11:02:10 AM »

Offline madeleine huey

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(SC.#1506;Gt-PECAM) & (B/D #550274; Rat-PECAM))
« Reply #16 on: February 27, 2006, 09:59:45 PM »
Hello,
Tried couple of time with 20 ug/ml Proteinase K/TE, 8.0 @ 37ēC for 15 min, then cool @ RT for 10 min for B/D Rat anti-PECAM (1:50) for overnight at room temperature, and 2nd antibody (Jackson Immuno
1:4000) for 1 hr @ RT, then finally (Jackson Immuno HRP-SP 1:500) for 1 hr.  There's no positive stain, except background.  
I also tried Santa Cruz Gt anti-PECAM with HIER (BioCare Reveal, ph 6,0) for 1 hr in BioCare pressure cooker.  Primary ab for Gt-PECAM at 1:50, 1:100, 1:200 for overnight @ RT with LSAB application.  They also don't work.  Background again.
Could you posted a more detail protocol for us.  For example, which vendor's 2nd & 3rd antibody did you use and for how long, and at which temperature for each individual antibodies.  
Desperate looking for Mouse Endothelial markers in FFPE.
Madeleine
650/467-1925
mhuey@gene.com

Offline richard03

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PECAM(CD31)IHC
« Reply #17 on: February 27, 2006, 10:09:14 PM »
I have tried this protocol and it works well. The key is to incubate proteinase K at 60 C instead of 37 C.

http://www.ihcworld.com/_protocols/antibody_protocols/cd31_pharmingen.htm

Offline madeleine huey

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PECAM(CD31)IHC
« Reply #18 on: February 27, 2006, 10:20:32 PM »
Thank-you Richard,
I will try your suggestion tomorrow.  By the way, where can I find your/any picture for the PECAM?
Madeleine

Offline richard03

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PECAM(CD31)IHC
« Reply #19 on: February 27, 2006, 10:30:34 PM »
This picture is from the same antibody but with zinc fixed tissue. Will post some pics from formalin fixed mouse tissue later.

http://www.ihcworld.com/imagegallery/
« Last Edit: October 21, 2007, 10:52:45 AM by richard03 »

Offline njcragg

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anti-mouse CD31
« Reply #20 on: February 28, 2006, 11:40:12 AM »
Just had to drop a post to say "THANK YOU" - you have just answered my prayers I have been trying to get this antibody to work for a couple of weeks.  I will be trying your protocol asap.

Nic
ic

Offline Linesider

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PECAM(CD31)IHC
« Reply #21 on: February 28, 2006, 02:43:46 PM »
There has been a number of protocols that work well for some people, and not for others, I see this over and over.
In our hands the antigen retrieval(cell marque , declere) for 60 minutes works routinely  on Zn/form and NBF tissue sections.
We have not had much sucess with the proteinase K protocol.
Primary santa cruz goat-mouse CD31(10 ug/ml), biotinylated anti-goat(jacksonImmuno, 1:800), followed by ABC elite.

Offline madeleine huey

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PECAM(CD31)IHC
« Reply #22 on: March 02, 2006, 07:42:00 PM »
Hello All,
I am having NO staining with MEC13.3 staining.  I used 20 ug/ml TE, ph 8.0 @ 60ēC for (25' & 30'), then cool @ RT for 20'.
Here's my protocol;
1.Deparaffinized & hydrate as usual
2.20 ug/ml Prot.K (Roche) @ 60ēC for 25 min and 30 min for another set.
Then cool @ RT for 20 min
3. Block with usual 2% NRS...../PBS, 7.2 for 30 min
4. Incubate 1st Ab; Rat anti-PECAM (1:50)/1%BSA.../PBS, 7.2) @ RT for overnight, then @ 37ēC for another hour
5. Incubate 2nd Ab; (Vector L) Bt. Rb-Rat (1:500)/PBS @ 37ēC for 30 min
6. Incubate 3rd Ab; (Vector L) HRP-SP (1:500)/PBS @ RT for 30 min
7. Chromagen AEC for 10 min
Result:  ALL NEGATIVE.  WHY???

Offline madeleine huey

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PECAM(CD31)IHC
« Reply #23 on: March 02, 2006, 07:45:44 PM »
Hello Linesider,
How long did you incubate your 2nd ab (Jackson Imm. Bt. Donkey- Rat, or Gt anti-Rat)?  Is it one hour @ RT?
Thanks!

Offline madeleine huey

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PECAM(CD31)IHC
« Reply #24 on: March 02, 2006, 07:50:25 PM »
Sorry Linesider, I type this too fast.  I forgot you are using S.C. goat anti-PECAM, so it should be Bt. Donkey-Gt/Ms-Gt. Did you also used pressure cooker for Declere, or water-bath for that one hour antigen retrieval as Richard posted in the PECAM protocol?

Offline Linesider

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PECAM(CD31)IHC
« Reply #25 on: March 03, 2006, 09:59:58 AM »
HI Madeleine-
We block with 10% donkey serum/TBS/1% triton-x, 20 minutes, at RT.
Declere antigen retrieval for 60 minutes in pressure coocker, cool 30 minutes.
Primary 60 minutes RT.
We use biotinylated donkey anti-goat, absorbed against murine, etc(1:800) for 30 minutes.
Followed by ABC elite 20 minutes.
All wash's in TBS for 5 minutes at RT.
All blocking/antibodies diluted in TBS/1% triton-X.
When we use the goat Isotype(Santa Cruz) we did see some cells taking up the antibody, but never persued, figuring out the cell type because they are easy to distinguish from endothelial cells.  More stringent blocking may eliminate this NSB but our theory was we finally had results, and we went with it.
Hope this helps.
bw

Offline madeleine huey

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PECAM(CD31)IHC
« Reply #26 on: March 03, 2006, 01:56:04 PM »
Hello Linesider/Richard,
Thank-you for all the inf. and replies.  
Madeleine

Offline njcragg

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anti-mouse CD31 (PECAM-1)
« Reply #27 on: March 07, 2006, 04:43:58 AM »
Still struggling to get any staining with the anti-mouse CD31, despite trying the protocol on the website.  Just used an ABC method though as haven't got a polymeric system for rat monoclonal.  Bought in a TSA kit from Perkin Elmer to amplify the ABC protocol - not tried it yet, but planning on doing it asap.  Has anyone got anymore suggestions?
ic

Offline Linesider

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PECAM(CD31)IHC
« Reply #28 on: March 07, 2006, 02:23:26 PM »
I would stay away from the tyramide, we too tried the amplification but the problem is not low expression of the PECAM-1 it is antigen accessibility, and the lack thereof.
Try the increased antigen retrieval protocol I outlined above, if it doesn't work you can send me one slide and I will stain it for you.

Offline njcragg

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anti-mse CD31 success
« Reply #29 on: March 16, 2006, 05:32:59 AM »
Finally got some staining with CD31, clone mec 13.3 and in fact with a fairly straightforward protocol.  Did the TSA and ran control (i.e. without TSA) at same time - got strong staining for first time but also got staining in control and so not due to TSA. Did a bit more investigation (realised the secondary previously used had gone off!!), tried a few more things and have come up with the following protocol :-

Endogenous peroxidase block before the enzyme digestion (think this is important, but will have to do further test to confirm)
Proteinase K for 5 minutes at room temperature (RT)
Primary at 1/50 overnight at 4degC
Biotinylated Rabbit-anti-Rat in 5% mouse serum 1/200 for 45 minutes at RT
ABC
DAB

Not sure about consistency yet, not very confident about this antibody and need to do yet another run but felt I should share this with you all, since you've been helping me.
ic

anti-mse CD31 success
« Reply #29 on: March 16, 2006, 05:32:59 AM »