RE: [Histonet] Background in frozen pancreas sections (continued...)

From:"Martin S."

 
Thanks Andrea,

My goat anti-rat is from Jackson (F(ab)2, mouse adsorbed) but I find
that if I don't dilute it in 10% normal mouse serum I get a lot of
background - diluted in this way it seems clean on spleen sections but
maybe its different for pancreas.
I used Vector's avidin/biotin blocking kit after serum block but before
primary - I will try this again concentrating on the washes.

Thanks again
Sonya



-----Original Message-----
From: Andrea Hooper [mailto:anh2006@med.cornell.edu] 
Sent: 19 February 2007 13:55
To: Martin S.
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Background in frozen pancreas sections
(continued...)

It's likely your secondary (anti-rat IgG) is cross reacting with mouse
IgG in the tissue. I would suggest you purchase an anti-rat IgG which is
highly cross adsorbed to many species INCLUDING mouse. This can be
purchased from Jackson ImmunoResearch for example.

Also, with pancreas you definitely need to do an AB block. You said it
increased background? That's odd and concerning ... how are you using
the kit? Lots of washes? I perform my AB block specifically after serum
block and before primary (with loads of washing) and am able to get very
little background.


----- Original Message -----
From: "Martin S." 
Date: Monday, February 19, 2007 5:28 am
Subject: [Histonet] Background in frozen pancreas sections
(continued...)

> Sorry just thought some additional info might be useful;
> 
> The pancreas tissue was frozen in OCT in a bath of isopentane on dry 
> ice, 10um sections were cut, air dried for 1hr, then fixed in acetone 
> for 10mins. I quenched endogenous peroxidase with Pierce Peroxidase 
> Suppressor.
> Sections without primary or secondary antibodies still have a littel 
> background but its worse with the secondary (I have tried rabbit 
> anti-rat:Biotin and it was worse).
> 
> Ta
> 
> 
> 
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