[Histonet] Re: Fluorescent antibodies for immunofluorescent staining and F4-80

gayle callis gayle.callis <@t> bresnan.net
Fri Sep 18 10:33:17 CDT 2009



You wrote (two separate messages) 


I've been given the challenge of finding a variety of fluorochrome

conjugated antibodies for IHC, however I'm having a great deal of trouble

finding them. We basically want a fluorescent primary antibody that can be

visualized instantly  without use of a secondary etc. 


We would like antibodies to bind to Osteocalcin, Alkaline Phosphatase,

Periostin, TRAP 5B, Cathepsin-K and N-Cadherin. 


If anyone has experience using fluorescent antibodies for the above or know

of anyone who produces them I would be most grateful if you could let me




I have recently stained for f4/80 macrophage marker on wax embedded mouse
tibia sections. I have managed to get staining however, I also have staining
on my negative control, which seems quite specific. 
I have investigated this and there seems to be no problems with the DAB and
all reagents used are exactly the same down to where they are bought as
those recommended in a protocol. 
If you have managed to stain for f4/80 on tibia sections without getting
staining on your control or you have any explanations for this I would much
appreciate hearing from you to compare protocols. 


For fluorochrome conjugated primary antibodies, you can conjugate these on
your own.  Have you done a search for these antibodies via Google?  

Be aware the some fluorophore conjugated primaries might not fluoresce IF
the antigens are in close proximity to each other.  The antibody binds but
the fluorochrome itself will not fluoresce due to a physical chemical
phenomena called quenching.  This is where the fluorophore trades electrons
instead, quenching any fluorescence you want to see.  If this happens , you
would have to detect the fluorophore with antibody, eg. antiFITC, anti Alexa
488 and so on.  You are back to square one then. Often it is better to just
detect the antibody with a secondary-conjugated to fluorophores, or buy a
biotinylated primary antibody and detect with Streptavidin Alexa dye
(fluorophore).   We do triple IF work for murine CD markers that does not
take days to perform, and double IF is very straightforward.  


As for the F4/80 antibody, you did not provide YOUR protocol so people can
respond to the question - fixation, decalcification,  retrieval, normal
serum blocking, primary and negative control concentrations, etc.   This
antibody works on FFPE tissue, and there are several postings on Histonet
(go to Archives) on protocols for successful staining for soft tissues.
The negative control should be negative though.  Did you do a dilution panel
to determine working concentration of primary?  What was secondary?   


Gayle M. Callis


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