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
> 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).
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