Immunohistochemistry - Technique Question

sa83164 at ODIN.MDACC.TMC.EDU sa83164 at ODIN.MDACC.TMC.EDU
Wed Mar 27 10:37:35 EST 1996


>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


When I do immunohistochemistry, I use the antibodies diluted in PBS/0.1%
BSA, even in the presence of other blocking agents like tween or triton. I
also usually include a previous blocking step using 10% serum in PBS/0.1%
BSA for 20'. Use serum from the species in which the secondary antibody was
made. Also, depending on your method of detection you may need to block
right after fixation (for example, if you detect by peroxidase staining,
you block with 0.3% hydrogen peroxide in methanol for 20'). One more thing,
if you are using archival tissue, some antigens may need an "antigen
retrieval" step if they have been fixed and then embedded in paraffin. This
is particular for each antigen and you have to try different techniques.

I hope this helps,

Ana Robles

sa83164 at odin.mdacc.tmc.edu





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