Immuno(histo)fluorescence: what's the best method?

Dr E. Buxbaum EB15 at
Thu Mar 14 10:07:10 EST 1996

Difficult to answer if you dont say what you want to do!

First: Are you using a monoklonal or polyclonal primary antibody? If you 
have a polyclonal antibody, you can use almost any fixative and embedd in 
parafin or PEG for cutting. If you use a monoclonal, this procedure may 
(or may not, trial and error required) block the antigen in just that 
position where the antibody binds. In this case you may have to use 

Second: What sensitivity do you require? Normaly a secondary antibody 
labeled with FITC or similar is used for fluorescence visualisation. 
Enzyme labeled antibodies with fluorescent substrates are more sensitive, 
but that goes along with higher backgrounds. 

Third: What is the required resolution? If you want to locate cells which 
contain your antigen, you can use enzymatic methods (fluorescence or 
metal-enhanced DAB). If you want to locate your antigen on subcellular 
structures, you have to use antibodies labeled with fluorescent dyes, as 
products of enzymatic reactions will spread out by diffusion.

By the way, there is no need to buy expensive kits for metal enhanced DAB 
staining! Simply include a few mM NiSO4 in the reaction mix. The brownish 
red reaction product of DAB with peroxidase is a strong reductant, and 
will precipitate elementary nickel. This is black (good contrast) and 
chemically stable enough to survive counterstaining and embedding in 
entellan, malinol or similar. Works good on western blotts too. 

Fourth: What is your equipment? If you have a nice fluorescence 
microscope at hand, you will want to use it.

Fifth: Do you want to do double labeling? If so, you are almost limited 
to fluorescence detection with dye labeled antibodies, you may even have 
to label your primary antibodies.

So, tell us what you want to do, if you need more help.

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