Double IFA staining per Re: [Histonet] RE: Double staining

Gayle Callis gcallis <@t>
Wed Mar 29 14:35:26 CST 2006

At 12:43 PM 3/29/2006, you wrote:
>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 

No, in fact you can mix two secondaries each conjugated to different 
fluorophores if you wanted to.

>I would question how the biotinylated component would cross react with the 

Bioinylated secondary cannot react with a fluorophore - it needs the next 
reagent, a Strepavidin-fluorophore (per the first question asked).

>...I have no direct experience to this.
>Then there's the dilution of each when they are placed together.

We merely make up one antibody and dilute the second in correct volume of 
the first primary dilution to have the correct concentration of our second 
primary. The diluent would be the same since the host of these two 
secondaries is identical (we would probably use 5% donkey serum).  Not a 
difficult task, and a little math.

>I see the main question is how well the flourophore can cohabitate with 
>your biotinylated secondary

No reason not to, however the main thing is to protect this step from light 
during incubation to prevent photobleaching of the fluorophore.

>Have you tried each stain separately?

An excellent suggestion and the best way to determine optimal results with 
EACH antibody and then combine in a cocktail for later staining (faster, to 
be sure!)

>Then doing the double stain the "traditional" way?

What do you mean by "traditional" way, a sequential staining?  Please 
elaborate with a protocol?   If you have not already done so, I strongly 
suggest you access Chris van der Loos's Immunoenzyme Multiple Staining 
Methods and see how cocktails of primaries and secondaries, etc can be 
cleverly used efficiently and without problems.  He is also presenting an 
all day indepth workshop on multiple staining at NSH in Phoenix this 

>If you could get a base line, that might help to see what happens when you 
>start making the voodoo brew.

We love our voo doo brews, see the following example and if my second 
primary antibody had been purified, I would have happily mixed the 
fluorophore conjugated secondary with a biotinylated secondary, and then 
the SA-Alexa dye.

Goat antiGFP in a cocktail with biotinylated CD11c (dendritic cell marker) 
rat antiMouse
Donkey anti Goat-FITC followed by
Strepavidin-Alexa 555 in a diluent that does NOT contain any normal serums 
- we cannot cocktail the Dk antiGoat FITC which we would dilute in donkey 
serum since SA can bind to any endogenous biotin in the normal serum (a 
warning given by Molecular Probes)

People doing FACS work are always making cocktails of their antibodies 
conjugated to fluorophores, sometime more than two or three without problems.

The only problem I ever had with mouse CD marker for an IFA stain was using 
a directly CD4-FITC - there was no fluorescence visible.  There is some 
physical barrier here, a spatial or stoichometric (sp?) 
problem/configuration that did not allow the FITC to glow.  It was 
sequestered in some way.

In our lab, we never use kits for double IFA work, it is all inhouse 
dilutions of stock/concentrated of primaries, secondaries, and Strepavidin 
Alexa dyes, something very typical with research labs at times.  The 
biggest problem with some of the kit secondary cocktails i.e. multilinks is 
that you don't end up with one of the links (mouse?) becoming a problem 
with background/unwanted cross reactions - something that would affect our 
mouse tissue work.

But it is sooo much fun

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

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