[Histonet] Re: Direct conjugates for immunofluorescence staining

[gayle callis]

Jennifer, 

 

You Wrote: 

I work with a scientist who insists on using primary antibodies directly

conjugated with FITC or Texas Red for IHC.  In my past experience these

directly conjugated antibodies didn't give a strong enough signal for

use in IHC (I've seen them used only for FACS analysis).  I would

appreciate your professional comments.

 

Jennifer M. Anderson, Scientist

Halozyme Therapeutics, Inc.

11404 Sorrento Valley Road

San Diego, CA 92121

858-704-8333

 

If your antibody is conjugated to a fluorophore, as you described, you may
run into the problem where the FITC and Texas Red molecules are in too close
proximation to each other and will "quench" due to a trading of electrons
between the FITC molecules when these are excited.  This is a problem we had
with murine CD4-FITC and murine CD8-FITC. The result is the antibody binds
to the antigen, a protein, but the quenching causes the FITC to not
fluoresce. Quenching is a NOT photobleaching aka "fading", but a different
physical chemical happening that "reduces the excited state lifetime, and
the quantum yield of the affected fluorophore."   You can go to
www.olumpusconfocal.com/theory/fluorphoresintro.html and read about this
under Quenching and Photobleaching (two different things). After long
discussions with my Physical Chemist husband who works with quenching
studies on fluorescent molecules, we do not use direct fluorophore
conjugated antibodies in the lab for staining tissue sections anymore. The
Jablonski Diagram also shows this.   

 

It will not happen with all antibodies that are conjugated to fluorophores
due to their spatial relationships (in terms of nanometers) but if it does,
Ray Koellings suggestion to come back with an antiFITC or Anti Texas red
should solve the problem. 

 

We are better served by having the primary antibodies biotinylated, then
come back with a Streptavidin Alexa fluor dye (488 for FITC, and 594 for
Texas Red).