[Histonet] Frozen sectioning human spleen

[gayle callis]

Dear Sonya, 

You need to find out how these people ARE snap freezing, then train them to
do it correctly. I have seen Histonet messages where people have success
freezing the way you described and had no problems or so it seemed although
I do not care for freezer freezing. If the liver is embedded in OCT in a
cryomold and the bottom of the cryomold is lowered slowly into LN2, they may
have better results. Just don't drop the mold into the LN2 or the OCT/tissue
may crack.  Freezer freezing at anytime due to slower freezing that creates
some huge ice crystal damage e.g. freeze artifact.  I will be happy to send
you an excellent discussion of freezing artifact from Dr. Charles Scouten if
you wish, found on the web. These people may be waiting too long before
freezing, or doing some funky little thing to create artifact. Whatever is
being done obviously is ruining the nuclei in center of sample but not the
edges where freezing is probably occurring much faster.  All artifact may
not be due to freezing either, although you have experience with murine
spleen.   
   
The thicker sections at 10 um could be pulling the nuclei out of the tissue
itself. Trimming too thick e.g. at a thickness exceeding 15 um or so, can
also do the same thing, especially at cold temperatures for this very
homogenous tissue.  Spleen contains a lot of blood, which doesn't section
well.  

Some suggestion for these problem these spleen samples.  If you already do
this, you can ignore or review: 

Correct snap freezing, although you can try this with the blocks from the
not exactly known freezing methods.

Set cryostat at -16C to -17C.  Warmer temperature allows for better
sectioning of homogenous spleen, also liver, brain, and spinal cord. 
Trim the block at thinner coarse setting.  Start trim with rapid coarse
advance until you get to the OCT, the trim into the tissue with fine
approx.15 um increments.  Do a final few turns of the flywheel at final 5 um
sectioning thickness.  This provides a smooth, clean block face with tissue
left intact, not with little chunks pulled out. 
Using a brand new disposable blade, section at 5 um.  I assume you use Plus
charge slides (you didn't say you used these?) 
To check that your sectioning is working well, do a quick Hematoxylin stain
on a section.  Pick up, fix with 95% ethanol, rinse, dip in hematoxylin 15
or more times, rinse, blue and coverslip.  Take a look at your morphology to
see if you have corrected the problem as nuclei should be intact. 
Continue with sectioning then proceed with your drying/fixation. 

Let us know if anything improves, and good luck on training the other labs. 

Good luck

Gayle M. Callis 
HTL/HT/MT(ASCP)  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James S.
Sent: Thursday, June 09, 2011 5:23 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Frozen sectioning human spleen

I've been trying to get some frozen sections of human spleen. Usually all
the work I do is with mouse tissue and I take the tissues myself and
immediately freeze in OCT in isopentane/dry ice. The problem with the human
samples is that I have no control over how they are frozen! I have one
sample from our path dept which was just put directly in the -80oC freezer
the other sample I have is from LN2 storage (I presume it was snap frozen in
LN2 after removal).

Anyway the sections from both samples are awful! They seem to cut ok but if
you do any staining on them they fall apart and any tissue left just doesn't
look right - the nuclei look misshapen, fuzzy, stretched and extracted. In
sections from the tissue stored in LN2 the nuclei were either missing or
didn't take up the dapi at all (except at the very edge of the section).

Has anyone had similar problems?

I cut 10um sections, air dry overnight, fix 100% acetone 10min.

Thanks
Sonya


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