[Histonet] IF Doublestaining

[andreahooper <@t> rocketmail.com]

I completely agree with all Adam has said. 

The essential key is to make sure your secondaries are highly cross adsorbed to each other and including mouse since you are staining mouse stroma. Also run each as singles alongside your double - that will give you great confidence in what you are seeing is real.

Should be a piece of cake ;)



Sent from my Verizon Wireless BlackBerry

-----Original Message-----
From: "Adam ." <anonwums1 <@t> gmail.com>
Date: Thu, 13 May 2010 13:43:57 
To: Igor Deyneko<igor.deyneko <@t> gmail.com>
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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