Re: Immuno on plastic

From:Geoff McAuliffe

    First is fixation, glutaraldehyde is a powerful cross-linker so you will
PROBABLY have to reduce the conc. of glut in the fix to 0.1 to 0.5 % and keep the
time very short, depending on many other factors like which antigen you are looking
for. A low pH-high pH paraformaldehyde sequence with just a bit of glut in the low
pH fix can give pretty good ultrastructure without killing (usually) too much
    Keep dehydration time to a minimum, do not store the tissue in alcohol. I have
heard that dehydration in graded acetones is better for antigenicity than graded
alcohols, but I have no evidence/references.
    Oh yea, forget post-fixation in osmium for the first trails.
    Epoxy. Epon (sorry, epon substitutes) or Araldite are pretty good, both respond
well to attempts to 'open' the epoxy for access by antibodies. Forget using
Spurr's, too highly cross-linked. There are some antigen friendly embedding media
for TEM but I know very little about them.
    Generally speaking, you can't get great ultrastructure AND great antigenicity
in the same tissue. Compromises will have to be made.
    A trip to the library/PubMed is in order here. Or hope someone who knows a lot
more answers!

marjorie lehman wrote:

> I have never done immuno on any plastic and now I have been asked to do it on a
> resin that can be used for TEM.
> The investigator wants to locate an area then cut it out to section for TEM.
> Oh boy, do I need lots of help here!!!   Please!!!
> Marge Lehman

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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