What is the major factor to affect the immunostaining of cytochrome C on embryo of ICR

Dr Engelbert Buxbaum eb15 at le.ac.uk
Fri Aug 14 12:49:53 EST 1998


Platini Kwok wrote:
 
> Recently, I was involved in the immunostaining of cytochrome c in embryo
> tissue. The tissue was fixed in 4% paraformaldehyde and dehydrated in
> graded alcohol, xylene and finally it was embared in wax block. It is
> found that only few positive signals were found in heart and bone tissue

The harsh treatment during tissue processing may or may not prevent
proper staining of an antigen. The risk is obviously greatest with
monoclonal antibodies, which have only a single binding epitope.
Polyclonal antibodies, with their multiple binding sites, are less
susceptible. 

There are several ways to control for this effect. One is to use frozen
sections from unfixed tissues instead of parafine embedding. This avoids
a lot of the denaturing processes. The second experiment you can perform
is to carefully trypsinize the sections before antibody exposure to
recover blocked antigenic sites. Works sometimes and sometimes not.
Briefly microwaving sections in citrate buffer has also been suggested
for this purpose. If you want to know only wether a particular antigen
is present in a particular tissue, rather than its subcellular
localisation, you may try immunoprecipitation of tissue homogenisates
instead of immunocytochemistry. Also, it may be worthwhile to do your
experiments with several different antibodies (there should be enough
available for a popular protein like cytochrome c).



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