Did anyone know whether the Epon-embedded nerve X-section tissue be re-used for immunocytochemistry

Jamie Boyd boydjd at ctrvax.vanderbilt.edu
Tue Sep 5 21:25:20 EST 1995


In article <41huf3$99k_002 at hkuxb.hku.hk>, email at xxx.xxx.xxx (fullname) wrote:

:
> (1) Did anyone know whether we can use those glutaldehyde/OsO4-fixed or
Epon-embedded sections for the immunocytochemistry? In other words, can
the epitopes on the nerve tissue/cell in X-section be still
preserved/unaffected or still be recognized by the respective Abs?

   Maybe yes, maybe no; it probably depends on the antigen of interest, as
some antigens are more robust than others. Also, different antibodies
towards the same target may show different sensitivities to fixation,
etc.  Usually, small molecules like amino acids (GABA, glutamate, e.g.)
can still be localized with antibodies following embedding. However, I
have seen occasional reports of peptides or even large proteins being
localized this way as well. 

> (2) If the epitopes were still remain unaffected, HOW can we remove the
Epon for exposing the epitope for immunocytochemical treatment?

Several different groups have published protocols for doing this. Here is
a reference to a well-known paper using post-embedding procedures to look
at GABA immunostaining in the electron microscope:

Somogyi, P. and Hodgson, A.J. (1985) Antisera to gamma aminobutyric
acid.III. Demonstration of GABA in conventional electron microscopic
sections of cat striate cortex. J. Histochem. Cytochem. 33:249-257.

Their procedure involved etching the resin with periodic acid and removing
the osmium with sodium periodate prior to incubating ultrathin sections in
anti-GABA, followed by a colloidal gold conjugated secondary antibody.

   Immunocytochemistry for light microscopy can also be done on tissue
prepared for Electron Microscopy and embedded in resin by cutting 1 to 5
micron thick sections, and etching the resin with ethanolic sodium
hydoxide. See:

Somogyi, P. et al. (1984) different populations of gabaergic neurons in
the visual cortex and hippocampus of cat contain somatostatin- or
cholecystokinin-immunoreactive material. J. Neurosci. 4:2590-2603  

Here, the standard peroxidase/DAB reaction was used for visualization.



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