[Histonet] DRGs

[Patsy Ruegg]

Carol,

I have had problems with cutting samples setup in histogel before but I
can't figure out why, the problem is inconsistent, sometimes the samples are
fine and other times it acts like the histogel prevented the infiltration of
processing reagents into the tissue.  I have even cut the histogel away (it
has done it's job of keeping the small tissue oriented) and reprocessed the
tissue with some good results.  Melt the block and cut the histogel away
with a razor blade, it just surrounds the tissue and does not really
infiltrate it, then gently squeeze the melted paraffin out while on a hot
embedding plate between paper towel, no need to remove paraffin with xylene,
just re cassette the tissue keeping track of the orientation and put it back
thru the processor.

Some theories I have for why histogel embedded samples cut well sometimes
and not others include:

1.  Maybe the histogel got too cold during the setup process, I try to make
sure the histogel is completely liquid (warmed) when I put it in the mold
with the tissue and then let them cool together, I think sometimes if I am
preparing a bunch of samples the last ones start to setup before I put the
tissue in the mold with the histogel, but I have not done a scientific study
of this. 

2.  I think that it might be better to go into formalin rather than 70%
alcohol after preparing the histogel embedded block as the alcohol may
harden the histogel too much too quickly preventing infiltration?  Again,
just a theory not tested in a controlled way??  Sometimes my samples are
already in alcohol and sometimes they are in formalin so I have been putting
them back into what they came in.

Regards,

Patsy 

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Barone,
Carol 
Sent: Thursday, February 18, 2010 11:24 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] DRGs
Importance: High

Histonetter's...we received a "boat-load" of mouse DRGs that had been
prepared in histogel and are cutting...well..not so good.
We normally do DRGs from FS and get beautiful results. 
 
We have used histogel before with other small sample and have never had
issues...  not sure if it is the Histogel or DRG's (fixed in 10% NBF and
then transferred to 70% before placed into the histogel).is the issue..I
seem to remember that histogel requires formalin and wonder if the
transfer to 70% is causing our problem ...but, obviously there is not
much room for error with such tiny- tiny samples and they are already
process and in paraffin? 
 
I am not quite sure how twe can improve the ones that came in histogel,
and were processed to paraffin a paraffin block....any idea's? any
experience? any anything? Thx- ASAP!
 
cbarone <@t> nemours.org
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