Immuno(histo)fluorescence: what's the best method?
Dr E. Buxbaum
EB15 at le.ac.uk
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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