[Histonet] Re: Histonet Digest, Vol 53, Issue 24

[AWeiss <@t> shorememorial.org]

Dear Histonetters!
I was wondering if anyone knows or has a good protocol for processing,
fixing, and embedding the pelleted cells. I was given a protocol by my
boss,
but I have a few questions about it.
Protocol I got:

Pellet cells, wash with PBS, re-pellet.

Remove PBS and gently add 10%NBF.

10NBF-until fixed 6-24 hours.

70-85-95-95-100-100-100% Ethanols(15-20 minutes each).

3 xylene changes 20 minutes each.

4 paraffin changes 30 minutes each(melted paraffin is the first heated step
60 degrees).

Embed and cut at 5 microns using a rotary microtome and accu edge blades,
lay out on a 47 degree water bath, let dry on a flat warming table or in
the
oven and they are ready for deparaffinization.

So I have questions as to how would i extract cellf from the test tube
without disturbing the pellet? Then what could I put them into for
dehydration with alcohols and clearance with xylene? As far as paraffin
goes
as well,are there any special cassettes designed for cells? Also as far as
embedding goes, would I do the standard embedding or are there special
techniques? I would appreciate any information/ protocols.

Thank you in advance.

Igor Deyneko

Infinity Pharmaceuticals,

Cambridge, MA

You can suspend the cells in histogel by surgipath and this will create a
envelope around the cells, you just need a small amt. to cover the cells.
Allow it to soldify and then release and process you would any other
tissue.
Hope this helps.


Andrea J Weiss BST CT (ASCP)
Cytotechnologist
609 653 3577 Ext 4907
aweiss <@t> shorememorial.org