RE: Immunos on Mouse tissue
I'll throw in my experience with Pharmingen's rat anti-mouse CD31. I got
good staining in formalin fixed paraffin sections after pretreatment with
DAKO's Proteinase K and diluting at 1:400 - 800. Detection was by standard
ABC with HRP/DAB. No need for TSA.
I also ran some paraffin embedded mouse sections fixed and either cold
acetone of zinc/tris. Vessel staining was even better than the FFPE
sections, and there was no need to use the Prot K. Contrary to what others
may say, I have had no problem with endogenous peroxidase when using
zinc/tris fixed tissues and HRP.
With 4% paraformaldehyde I would suspect you need to play with the
Proteinase K pretreatment times, which will be dependent on the total
fixation time. In parallel, if the tissues were fixed a long time HIER would
probably be worth a try.
Brett M. Connolly, Ph.D.
Merck Research Laboratories
Dept. of Neuroscience
PO Box 4
West Point, PA 19486
From: Luis Chiriboga [mailto:Luis.Chiriboga@med.nyu.edu]
Sent: Thursday, January 31, 2002 1:56 PM
To: Barbara Wright
Cc: Histonet; Donella.Stillings@UCHSC.edu
Subject: RE: Immunos on Mouse tissue
I am curious, I have been unsuccessful with CD31 from Pharmigen. Trying to
use it for MVD study on mouse brain fixed in paraformaldehyde. Do you HIER.
Do you digest the tissue (I think recommended)? What dilution do you use? I
presume that this is the rat primary. Do you really need Tyramide Signal
From: Barbara Wright [mailto:bwright@gene.COM]
Sent: Thursday, January 31, 2002 12:40 PM
Subject: Re: Immunos on Mouse tissue
If you are using the DAKO CD31 antibody - it will not react with mouse
endothelium. Pharmigen CD31 (MEC13.3) works well on FFPE with Vectastain
ABC followed by NEN TSA-kit. And our Collagen IV is from a polyclonal from
Chemicon. Both work well on FFPE mouse tissues.
> I am in need of some help? I am trying to do immunos on mouse tissue. I
> currently use the Dako LSAB+ kit and I know that some of my antibodies are
> monoclonal. I have tried the Dako ARK kit and have not had any luck. I
> know what I may have done wrong. I also have tried the Dako Envision
> polyclonal kit. I have changed my antibodies from mono to poly or goat. I
> have tried different retrieval methods. What can I do now? Any help would
> appreciated. The two antibodies I am working with mostly is the Cd31 (goat
> polyclonal and Rat anti mouse) and Collagen 4 (monoclonal and polyclonal).
> Donella Stillings
> University of Colorado HSC
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