cytochemistry : fixation?!

Dr E. Buxbaum EB15 at le.ac.uk
Thu Oct 23 17:06:52 EST 1997


KAREN TILMANT wrote:

> Usually I'm doing imunohistochemistry but now I want to know how I've to
> prepare my cells (coated glass-slides, fixation, etc.) for
> immunocytochemistry?!

Coat cover slips with poly-lysine. Trypsinize your cells as usual and
plate out in a petri dish with the cover slips. Let the cells grow a
couple of days, until you have a confluent layer. Briefly wash with PBS,
then you can fix them any way you like, by just dipping the slips into
the solution. Formaldehyde in PBS is frequently used (4-10% for a few
minutes, a question of trial and error). 

> And does anyone know if it's possible to prepare a "smear" (or something
> like that) of bacteria, so that it's possible to stain them with the
> principles of immunohistochemistry?

You can smear a drop of bacteria suspension like you would prepare a
blood smear.  Then air dry. For fixation simply move the slide a few
times through the flame of a bunsen lamp, until the slide is to hot to
be touched comfortably (but not hotter! The aim is fixation, not the
production of charcoal). Should this interfere with antibody binding,
other methods of fixation (for example neat methanol) could be tried.



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