[Histonet] IF Doublestaining

[Beckham, Sharon]

Yes, I do double staining very frequently the way  Adam stated.  I hardly ever have to do the sequential staining.
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Adam .
Sent: Thursday, May 13, 2010 1:44 PM
To: Igor Deyneko
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] IF Doublestaining

In my experience, most fluorophores are quite stable to many washes. Here is what I would try at first:

1) Stain with rat anti-mouse and rabbit anti-human overnight at 4C in a humidified chamber. Dilute each antibody in a single volume of your favorite staining buffer (PBS, TBS, TBS-T). For example, if your rat antibody needs to be diluted in 1:100 and your rabbit needs to be diluted 1:200, take an aliquot of 200 uL of buffer and add 2 uL of the rat primary and 1 uL of the rabbit. Add the appropriate volume (usually 50-200 uL) of that mixture to each slide.

2) Wash the next day, and add your secondaries again to a single mixture for
1 hour at room temperature. As long as your secondaries have been highly cross adsorbed to many species, you shouldn't have a problem.

3) Wash, counterstain, mount, enjoy.

I really don't think you need to do sequential staining for this. I've heard anecdotal reports of two primaries or two secondaries forming immune complexes when mixed together, but I've never really had that problem.

Good luck,
Adam

On Thu, May 13, 2010 at 1:00 PM, Igor Deyneko <igor.deyneko <@t> gmail.com>wrote:

> Hello Everyone!
> I'm planning to try some IF co-staining with 2 antibodies, one is a 
> rat-anti-mouse and the other one is rabbit anti-human on a  xenograft, 
> each has an appropriate secondary, donkey anti rat and donkey 
> anti-rabbit, conjugated to 488 and 593. Can someone advise the best 
> way to perform such a procedure, I'm afraid the rules of sequential 
> staining might not work due to fluorophore instability with washes. if 
> anyone has performed such type of stain, i would appreciate any tips.
> Thank you.
> Igor Deyneko
> Infinity Pharmaceuticals
> Cambridge, MA 02139
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