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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of James S. Sent: Thursday, June 09, 2011 5:23 AM To: 'histonet@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 ------------------------------------------------------------ _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet __________ Information from ESET Smart Security, version of virus signature database 6191 (20110608) __________ The message was checked by ESET Smart Security. http://www.eset.com __________ Information from ESET Smart Security, version of virus signature database 6191 (20110608) __________ The message was checked by ESET Smart Security. http://www.eset.com __________ Information from ESET Smart Security, version of virus signature database 6191 (20110608) __________ The message was checked by ESET Smart Security. http://www.eset.com _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet