Re: [Histonet] blood vessel staining?

From:Jackie.O'Connor@abbott.com

I routinely perform IHC for microvessle density using Pharmingen's PECAM 
antibody using Streck fixed tissues in paraffin, 5 or 6 micron sections.  
Pharmingen includes an SABC protocol with their antibody that works 
extremely well - I use the antibody at a 1:30 dilution overnight at 4 
degrees C with no antigen retrieval.  It's the best I've found so far. 

Jacqueline M. O'Connor HT(ASCP)
Abbott Laboratories
Global Pharmaceutical Research and Development
Discovery Chemotheraputics





"rocan@mac.com" 
        cc:     "Stylli, Stanley" 
        Subject:        Re: [Histonet] blood vessel staining?


Stan,
I avoid antigen retrieval like the plague. Therefore I would recommend 
to not use the formalin in the first place if at all possible.   I 
believe that for formalin fixed samples my tech used citrate but I am 
not sure (long time ago).  Also, in general we are having very good 
luck with the L.A.B. antigen retrieval solution that was mentioned here 
a few months ago.
  We normally use use Streck's fixative when we plan to use the 
pharmigen anti-cd31 antibody on certain samples.  We also have used the 
antibody without antigen retrieval when we fix by intracardiac 
perfusion with 4% paraformaldehyde.  Doing the intracardiac perfusion 
with 4%PFA actually fixes the vessels before they collapse.
I think the key for this antibody is thick sections and long incubation 
at room temperature.  The thicker the sections the more you can 
actually visualize all intact vessel including capillaries.  The 
branching is clearly distinct, beautiful!

Rocio

On Mar 23, 2004, at 4:20 PM, Stylli, Stanley wrote:

> Rocio
> WHat sort of antigen retrieval have you used ?
> Has it been successful ?
> thanks
> Stan
>
> -----Original Message-----
> From: rocan@mac.com [mailto:rocan@mac.com]
> Sent: Wednesday, 24 March 2004 11:02 AM
> To: 'histonet@pathology.swmed.edu'
> Cc: Sylvia Poulos
> Subject: Re: [Histonet] blood vessel staining?
>
>
> Sylvia,
> The Pharmigen antibody works great but you may have to do some antigen
> exposure. We have found that the best staining is on thick frozen
> sections(20um) and we leave a 1:100 dilution incubating overnight AT
> ROOM TEMPERATURE!
> Our secondary is an anti-rat biotinylated (I think this one is from
> Biomeda).  We then use the ABC kit and DAB from vector.
> Good Luck
> Rocio
>
> On Mar 23, 2004, at 2:46 PM, Sylvia Poulos wrote:
>
>> Hi all,
>> I've got some formalin fixed paraffin embedded mouse tissue.  Any idea
>> of something I could do to stain capillaries in these?  Someone here
>> mentioned mason's trichrome.  Any thoughts are always welcome. Thanks!
>> Sylvia
>>
>> Sylvia P. Poulos
>> USDA-ARS-Animal Physiology Research Unit
>> Athens, GA 30605
>> 706-583-8279
>> 706-542-0399 (fax)
>>
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>
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