[Histonet] RE: Double staining

Guillermo Palao gpbnas <@t> yahoo.es
Fri Mar 31 09:06:17 CST 2006

My two cents is just to follow Andrea's advice on using pre-adsorbed secondaries (I also use Jackson's with great results), it will really save you a lot of time wasted on optimizating conditions.

"Andrea T. Hooper" <anh2006 <@t> med.cornell.edu> escribió:
  Hi Anna,

Your experiment will work! We do this all the time here with very 
nice results and it also sounds like you know what you are talking 

I suggest you check to make sure the secondaries are highly cross 
adsorbed and if not, buy ones which are (Jackson ImmunoResearch sells 
awesome secondaries).

I also strongly suggest you run each independently to make sure 
everything is in order. I normally do this alongside my double stains 
each time.

Also as Chris suggested you can even incubate your primaries together 
making your experiment even additionally streamlined.

And of course remember to block appropriately.

Good luck,

>I am very interested in this.
>I am not a chemist, so I would welcome more educated predictions.
>But theoretically, this secondary cocktail MIGHT work. My exposure to
>this is very limited and is based on my experience with the Biocare
>doublestain secondary cocktails.
>You would need to get more information on the actual chemistry of the
>secondary components...would the flourophore "smother" out the other
>secondary? I would question how the biotinylated component would cross
>react with the flourophore...I have no direct experience to this.
>Then there's the dilution of each when they are placed together...
>I see the main question is how well the flourophore can cohabitate with
>your biotinylated secondary
>Have you tried each stain separately?
>Then doing the double stain the "traditional" way?
>If you could get a base line, that might help to see what happens when
>you start making the voodoo brew.
>Let me know how this goes!
> Becky Orr CLA,HT(ASCP)
>IHC Lead
>Evanston Northwestern Healthcare
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu


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