[Histonet] Isotype background

[Margaryan, Naira]

Hi Adam,

I always do the isotype control parallel with a no-primary control in parallel with my real Ab. 

May I suggest you to use Protein block serum free and pure Ab diluent without adding 2% donkey serum?

Try this and let me know your results.

If histonetters think I am wrong, fill free and please, let me know. 

All the best,
Naira 

-----Original Message----------------------------------

Message: 11
Date: Tue, 13 Oct 2009 16:48:46 -0500
From: "Adam ." <anonwums1 <@t> gmail.com>
Subject: [Histonet] Isotype background
To: histonet <@t> lists.utsouthwestern.edu
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Hi all,

I am trying some IHC, and I am having a peculiar problem. Like I expect, my
antibody of interest (anti-mouse goat polyclonal) stains nonspecifically at
high concentrations (10 ug / ml) but as I titer it down (3 ug / mL), it
seems to stain relatively specifically the cells I think it should stain.
However, at 3 ug / mL, my isotype goat IgG stains nearly everything.

Here is my protocol
1) Block in 3% H2O2 for 10'. Wash.
2) Block in 10% donkey serum for 1 hr. Wash.
3) Block in avidin 15', wash, biotin 15', wash (Vector Labs blocking kits)
4) Incubate with primary / isotype overnight at 4C in 2% donkey serum. Wash.
5) Incubate with secondary (biotinylated donkey anti-goat, cross adsorbed to
mouse) in 2% donkey serum for 1 hr at room temp. Wash.
6) Incubate with strepavidin HRP in TBS-T for 30'. Wash.
7) Incubate with DAB+ (Dako) for 5'.

For isotype, I am using Chrompure Goat IgG, whole molecule from Jackson.
Some people have suggested that I just do away with isotypes altogether and
use a no primary control instead. I think there is some merit to this idea,
but I still think my issue might be indicative of a larger technical problem
in my staining protocol.

Thanks,
Adam