[Histonet] Pericyte, smooth muscle cell IHC problems

[Melissa Gonzalez]

Thank God! I thought I was going crazy! When I found Santa Cruz's I was thrilled because it worked on everything and looked great. Then it suddenly went dead no matter what I try it on.  I hate it when these things happen. 
FYI- R&D systems rat x mouse CD105 works really well, as does Serotec's CD34. 
But I know some people insist on Pecam-1 as being the most specific and reliable for blood vessels, and now I have no product to use. 
 
Melissa

-----Original Message-----
From: Jackie M O'Connor [mailto:Jackie.O'Connor <@t> abbott.com]
Sent: Thursday, May 05, 2005 11:03 AM
To: Melissa Gonzalez
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Pericyte, smooth muscle cell IHC problems



HEY!  I've had the same problem with the Santa Cruz CD31!  How utterly frustrating!  I had a lot from 11/04 that didn't work, and when I requested a replacement this month, that didn't work either!!  I've really come to depend on that antibody for microvessel density in my xenografts.  I haven't been able to find a comparable CD31 to work in FFPE mouse.  The BD will work on zinc fixed, tho, without antigen retrieval.   Anyone else have any tidbits for FFPE murine tissue? 

Jackie O' 
Jacqueline M. O'Connor HT(ASCP)
Assistant Scientist
GPRD Cancer Research
Abbott Laboratories, Abbott Park, IL
Jackie.OConnor <@t> abbott.com 



	"Melissa Gonzalez" <Melissa.Gonzalez <@t> cellgenesys.com> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu 


05/05/2005 12:19 PM 


        
        To:        <histonet <@t> lists.utsouthwestern.edu> 
        cc:         
        Subject:        [Histonet] Pericyte, smooth muscle cell IHC problems



Dear all, 
I have been trying unsuccessfully to stain for pericytes/ smooth muscle cells in sub cutaneous gliomas in a mouse model. (Tissues are either 4% PFA perfused or 10% FFPE)
I have tried several products, including:
* NG2 (Chemicon): works nicely in normal mouse organs, but binds to much of the tissue in the gliomas, making it impossible to interpret. 
*PDGFRB (Santa Cruz): resulted in a few crazy-appearing vascular areas, very weird
* ASMA (DAKO, Sigma, US Bio, Neomarkers, Abcam)
*Desmin (DAKO, Santa Cruz)
--in both monoclonals (using a mouse on mouse kit which did not work) and polyclonals, for which I only see staining in the attached skin and a very rare area in the tissue. I have tried both immunofluorescence on the perfused,  and chromogenic detection on paraffin sections, using Citrate, high pH, pepsin, and proteinase K retrievals. 

Is it possible that smooth muscle cells would not be well developed around blood vessels in a sub cutaneous glioma, and that is the reason I only see staining in the attached skin with a rare occurrence in the tumor? (Tumors are very vascular, blood vessels are definitely present)

Also, what is everyone using for blood vessel markers? We used to use BD Pharm. Pecam-1, which doesn't work anymore on anything except frozens, Santa Cruz goat Pecam-1 was working great but the most recent lots I ordered now appear completely negative using any detection. I have now switched to using CD105.

Any help would be greatly appreciated as I have tried all the tricks I know, and am left completely puzzled. 

Thanks so much, 
Melissa


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