[Histonet] CD31/PECAM-1

[Joanne Mauger]

To Isaiah'
I used CD31 from Pharmingen also, about 6 years ago. I used a 1:50 dilution after retreieval with pepsin for 30-40 min. at 37C(depending on length of fixation.
If the tumors are human cells in mice, the Dako anti human CD31 will probably work at about the same dilution, with pepsin as well. 
Jo

>>> "Isaiah G. Schauer" <is135475 <@t> bcm.tmc.edu> 03/27/04 08:41PM >>>
Histonetters,

Our lab has used Pharmingen's CD31 antibody (cat# 01951A/D) with great 
results for a couple years for paraffin-embedded mouse tissue IHC. 
Recently, they decided to "sublet" the production of this antibody to a 
company called Cymbus, which sells the antibody at 1/10th the original 
protein concentration. We've called Pharmingen regarding this, and they 
staunchly maintain that there is no difference between their antibody 
and the Cymbus rendition.

We now have to use a 1:5 dilution, on mouse xenograft tumors fixed in 
4% paraformaldehyde 16 hrs at 4C. The cost of repeatedly using this Ab 
has now become prohibitive for the lab.

Can anyone recommend a better CD31/PECAM-1 antibody for mouse xenograft 
IHC on 4% PF-fixed, paraffin-embedded tissue? Histonet archive searches 
have turned up Santa Cruz (cat# sc-1506), Dako (cat# M0823, clone 
JC-70A) and Serotec (cat# MCA1334G) as possible suppliers, but I wanted 
to hear from someone with paraformaldehyde-fixed, mouse tissue 
experience.

We don't currently use enzymatic digestion prior to staining, but I've 
read on Histonet of people using proteinase K, pronase or trypsin at 
varying temperatures for varying times for CD31 staining. Might anyone 
recommend a starting time/temp for my particular tissue type/fixation 
that could allow me to increase the 1:5 dilution of the Pharmingen 
antibody?

Thanks for your help and time.

Sincerely,
Isaiah Schauer

3rd year graduate student
Lab of David R. Rowley, Ph.D.
Baylor College of Medicine
Department of Molecular and Cellular Biology
One Baylor Plaza, room 326D
Houston, TX 77030

lab: 713-798-6221
fax: 713-790-1275


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