Auditorium wrote:
>> I'm doing some immunohistochemistry on paraformaldehyde fixed specimens.
> I have a question on the reagent that the primary and secondary antibodies
> are made in. These antibodies are from Peninsula Laboratories (they are
> not on the Web, I've done a Lycos search). The package insert says to mix
> these antibodies in a PBS/Triton-X solution. There is not a mention of
> including a non-specific protein (such as non-fat milk or normal goat
> serum or BSA, etc.). I'm curious as to why most of the other techniques
> using a primary and secondary antibody to detect a protein require that
> both of the antibodies to be mixed up in a fairly high concentration of a
> non-specific protein. I've followed Peninsula's directions and my
> specimens had very poor signal to noise ratio. Could it be that I should
> have made these antibodies in a protein solution? Email me back at
>tcc5g at virginia.edu.>> Thanks
>> T. Chai
T. Chai,
Typically we dilute the primary (and the secondary) in a solution of PBS/ 0.3% Triton
x-100/ 2% normal serum. The normal serum is from the animal in which the secondary is
made. You can add 1% BSA if you wish but this does not always help. Background depends on
several things. Sufficient PBS rinses between steps is very important. Excessively high
concentrations of primary can raise background. You don't say what method you are using to
visualize the antigen/antibody complexes in you tissue. If you are using peroxidase methods
then pretreating sections with 0.3% H2O2/PBS for 15-20 minutes is very helpful in
eliminating endogenase peroxidase activity which can be a major source of non-specific
staining. We use vector labs ABC elite kit to visualize results and it has proven very
succesful. It is based on the peroxidase method but enhances the signal. You must use
biotinylated secondaries which are included in the respective kits (as well as normal sera
too) If you are using fluorescence techniques then washes are extremely important both
between incubations and after the secondary. You may have to tinker with the concentration
of the secondary a little (this might help with the peroxidase method too if you don't have
a secondary that is clean, i.e. cross reacts with Ig's of the species from which your
sample came.) There are a lot of possibilities for non-specific signal but these are
pretty common causes and might help. I recommend a book by Strickberger on
Immunohisotchemistry (don't remember the exact title) which discusses in detail these and
other possible problems. Good luck with your experiments!
--
Jerry Clayton
jerry.clayton at UCHSC.edu
Dept. of Neurology
University of Colorado Health Sciences Center
Denver, Colorado USA
