Double IFA staining per Re: [Histonet] RE: Double staining
gcallis <@t> montana.edu
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
>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
>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)
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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