[Histonet] sample woes

Patsy Ruegg pruegg <@t> ihctech.net
Sat Jan 21 10:39:03 CST 2012

Please advise.


I was sent a bunch of sample by an investigator who is not too sharp. The
samples were prepared thus:


Pancreases were removed, washed placed in a modified Zamboni fixative (2%
formaldehyde, 15%Picric acid in 0.1M PBS, pH 7.5). Sample portions were cut
from the head, body and tail of the pancreas in 3 mm by 3 mm sections.

The pancreatic tissue samples from the various regions of the pancreas were
fixed overnight; samples were equilibrated in 50% sucrose in 0.01M PBS for
12 hours at 4°C, and mounted in Tissue Tek OCT Compound (Miles Inc.); 0.015
mm (15 μm) thick sections were obtained by cryostat sectioning. 

SAMPLES WERE FROZEN IN 50% Tissue Tek 50% sucrose , then shipped on wet ice,
not dry ice.

These frozen tissue samples were in small embedding molds with a tiny dab of
the OCT/sucrose gel on top of them, it looked like none was under them. They
shipped these supposedly previous frozen samples to me on wet ice, not dry
ice (dumb move), the molds were open on top in a box, the sample thawed and
the gel and sample was leaking out of their molds, it was a mess. The
investigator who sent the samples to me said this "the samples arrived
within 18hrs as signed for, so they should have still been frozen", how this
person got to be the CEO of a Biotech company I cannot imagine. 


They wanted me to just refreeze them and cryosection them to test some abs
they sent with them, I said that I would not do that, I did agree to take 2
samples as a pilot study and fix them in formalin overnight and then process
and embed them in paraffin, which I have done. If I can get them to section
(they are still soft and squishy ) I was planning on doing an H&E stain and
look at the samples to see if they look preserved at all. My question to you
all is this: if the tissue looks viable by H&E what should I try to test
immuno reactivity viability? I was thinking of running an antibody like
vimentin or cytokeratin or something, but these are ms pancreas so maybe I
should use insulin ab or something else. 


Thank you for your valuable advise,






Patsy Ruegg, HT(ASCP)QIHC


12635 Montview Blvd. Ste.215

Aurora, CO 80045


fax 720-859-4110




More information about the Histonet mailing list