[Histonet] Background in frozen pancreas sections

[Martin S.]

Thanks Joost, I dilute my secondary in normal mouse serum (10%) and it
has decreased the background but have not tried pre-incubating the
secondary for 1hr as you suggest - will try doing this today!

Thanks
Sonya

-----Original Message-----
From: Bruijntjes, J.P. (Joost) [mailto:joost.bruijntjes <@t> tno.nl] 
Sent: 19 February 2007 10:34
To: Martin S.
Subject: RE: [Histonet] Background in frozen pancreas sections

Hi Sonya

My two cents: have you ever tried to reduced the background staining by
preincubating your second antibody with normal mouse serum. I had
similar problems with rat tissues incubating them with monoclonal
(mouse) antibodies. But there is a strong similarity between the rat and
mouse immunoglobulins. When I preincubate my secondary step for 1 hour
with a certain amount of rat serum the background is vanished, and my
lymphocytes are beautiful. 

Joost 
 
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Martin
S.
Sent: maandag 19 februari 2007 11:06
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Background in frozen pancreas sections

Hi All,
 
I've been doing some CD8 staining of frozen sections of mouse pancreas
with autoimmune insulitis. The CD8 staining is good (using rat antiCD8 +
goat anti rat:Biotin + ABC/HRP + DAB) but there is high background
staining. The problem is I get a lot of brown/grey background in the
exocrine cells but the islets are completely clean.
I've tried numerous different blocks containing BSA, goat serum and/or
mouse serum and/or Tween I've also tried using avidin/biotin block
(Vector), this actually increased the background.
 
Any suggestions?
 
 
Thanks
Sonya
 


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