Re: [Histonet] double/triple staining with streptavidin-Alexa Fluor

From:Gayle Callis

Yes, however, using an avidin/biotin blocks before each antibody to ensure 
1) quenching endog biotin before first and quenching any residual biotin 
left over from first antibody sequence before you do the second primary 

We do double IFA staining with two rat antimouse monoclonals in the 
following way with one purified monoclonal and one biotinylated monoclonal 

Frozen sections fixed with acetone alcohol mixture at RT after overnight drying
Normal serum block matched to host of secondary used
Strepavidin/biotin block (Vector)
Rat antiMouse monoclonal
Donkey antiRat F(ab')2 frag of IgG conjugated to RRX
Rat serum block, 5% for 15 minutes
Primary #2 rat antiMouse-biotinylated
Strepavidin-Alexa 488
Prolong gold antifade with OR without DAPI

If we do something like an Goat anti GFP/Donkey antigoat-FITC and Rat 
antiMouse CD11c-biotinylated/Strepavidin-Alexa 555, we shorten our protocol 
time considerably.  A cocktail of Goat antiGFP and Rat antiMouse CD11c, 
followed by Dk antiGoat secondary-FITC, and then SA-488.   This gets rid of 
so much sequential staining and blocking because that is all done in the 
beginning with Normal serum and the avidin/biotin blocking.

To do this, "Will sequential staining (i.e. first anti-rabbit and SA488, 
then anti-mouse and SA555) saturate all biotin binding sites from the first 
step ?"  I would advise doing either Strepavidin/biotin block before each 
primary (one could use Avidin/biotin block) particularly before the second 
primary to ensure saturation of biotin binding sites.

We prefer to do a secondary conjugated with fluorophore for one of the 
antibodies, and make sure your secondaries ARE adsorbed to the species 
being stained and do NOT cross react in any way.

Some people have success using secondaries conjugated to Alexa fluorophores 
(you can do your own conjugations with their kits) and not end up with so 
much Strepavidin fluorophores in the protocol which makes for a LONG day of 

At 01:25 PM 3/14/2006, you wrote:
>Dear HistoNetters,
>Is there a way to perform double (or triple) staining using secondary, 
>biotinylated antibodies with streptavidin-Alexa Fluor conjugates, for 
>instance, anti-rabbit, biotinylated secondary antibody A, detected with 
>streptavidin-AF488 in combination with anti-mouse, biotinylated secondary 
>antibody B, detected with streptavidin-AF555, preventing cross-reaction ? 
>Will sequential staining (i.e. first anti-rabbit and SA488, then 
>anti-mouse and SA555) saturate all biotin binding sites from the first step ?
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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