RE: [Histonet] Fixing & Processing Cell Pellets

From:sheila adey

We purchase a cyto block kit from Thermo Shandon. Works great.Sheila Adey HT MLTPort Huron HospitalMichigan> Date: Wed, 16 Apr 2008 11:02:52 -0400> From:> To:;> Subject: RE: [Histonet] Fixing & Processing Cell Pellets> CC: > > We used to use Histo-gel from Richard Allen, it worked quite nicely,> kept the pellet together and processed well. It didn't interfere with> any IHC either.> > I'm sorry that I can not give you more specifics as I am currently in a> new position without our protocol in from of me and do not want to give> you any misinformation, but I believe that after the fixation, we> followed the manufacturer's protocol. Here is the link:>> > > > Angela McNabola, MS HT(ASCP)SLS, QIHC> Research Scientist> Ikonisys, Inc.> 5 Science Park> New Haven, CT 06511> 203-776-0791>> > -----Original Message-----> From:> [] On Behalf Of Igor> Deyneko> Sent: Wednesday, April 16, 2008 10:45 AM> To:> Subject: [Histonet] Fixing & Processing Cell Pellets> > 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> _______________________________________________> Histonet mailing list>>> > _______________________________________________> Histonet mailing list>>
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