fixing for ihc

[Diana van Driel]

>This is a question for people who routinely do immunohistochemistry.  I
>am wandering about the degree of fixation necessary to achieve the best
>results.  Is there a danger of "over-fixing"?  I perfuse rats with about
>300 ml of 4% paraformaldehyde and 0.025% glutaraldehyde over 6-7 min.  If
>you have a better protocol, please reply.  Thanks.
>
There is insufficient information in your question. What antigens are you
looking at? What antibodies are you using? Some antigens survive anything;
eg GFAP is happy with 2.5% glutaraldehyde, osmium and resin embedding.
Others don't even like paraformaldehyde. Some antibodies are designed for
use with unfixed tissue, some for paraformaldehyde fixed tissue, yet others
for glutaraldehyde fixed tissue. What visualisation system are you using?
-some are more sensitive than others. I suggest you read the literature on
your own antigen/antibody/detection system and then post a more specific
query.


Diana van Driel
Dept Ophthalmology
Sydney University
AUSTRALIA 2006