Good afternoon, I am having a few issues getting nice morphology from both liver and heart tissues. I currently work only with CNS tissues (brain, spinal cord, DRG, etc.) but recently, our research team have become interested in immunostaining some peripheral tissues, including the heart and liver.
All tissue [mouse] is perfusion fixed with 4% PFA and then post-fixed in fresh 4% PFA for at least 24 - 48 hours. After fixation, the tissue is cryoprotected in 30% sucrose solution for 48 hours. Once cryoprotected, I then section the tissue at various thicknesses depending on the assay I'm running. Previously I sectioned some heart and liver samples (sectioned both at 20um and at 8um) and ran a few H&Es. Unfortunately, the tissue was so damaged and under-fixed that we scrapped the blocks. This time around, the mouse liver tissue was carefully dissected prior to post-fixation into four quadrants to allow for better PFA permealization. Additionally, we post fixed in 4% PFA for 3 days instead of 2, and the tissue was cryoprotected for 2 days. To my surprise, when I sectioned this tissue on the cryostat, I still noticed severe artifacts. It is very difficult to see nice morphology, and there appears that there was an issue with fixation (in the liver the nucleus is flattened and the sinusoids are not clear, etc. the nuclei in the heart are also more flat and the muscle fibers have separated from one another). The staining was not as bad as the first tissue I had sectioned, but I was still unable to get a nice H&E stain depicting clear nuclear/cytoplasm. These artifacts appeared at both 8um and 20um. Does anyone happen to have nice fixation/H&E staining protocols for both liver and heart? I'd be happy to give an in-depth description of the protocol(s) I'm currently using. Additionally, is there anything that appears I'm doing wrong in terms of perfusion/fixation? Thanks so much! Allyse _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet