Hello Histonet!
I am hoping that someone could shed some light on some issues I am having with some human knee tissues embedded in paraffin. About one year ago, we started having some issues with the established protocol and decided to increase the processing times. Issues we are seeing: Once processed, the stained ligaments (MCL, LCL, ACL, etc) appear to have brittle tissue (sort of wavy looking) toward the middle of the sample in stained sections. The block itself has white brittle areas that flake off. Regarding bone slabs, we find that the junction between the cartilage and bone is where the tissues are the hardest after decal and the color is different in this area. We think that the fixation may be a problem because of penetration. If you have any suggestions for ensuring complete fixation, that would be very helpful! The processor run does not include a fixative station. Soft Tissues and Ligaments: All tissues are trimmed down to about 4mm in thickness and put into Anatech’s Z-fix for 5 days with one change of Z-fix. Washed and put into 70% EtOH. Bone: Slabs are 6-9mm thick (we ideally aim for 7mm) across the tibial plateau and along the medial and lateral femoral condyles – with cartilage and about 1cm of subchondral bone. The slabs are individually fixed with Z-fix for 1 week, with one change of Z-fix and excess bone trimmed after the first 3 days. Then the samples are put into TBD-2 (formic acid decal) for up to 2 weeks depending on the end point, with a change of decal. Once decalcified, the samples are cut into 20mm X 4mm X 7mm pieces and washed and put into 70% EtOH. The processor completes all steps under vacuum. Older Protocol with another processor: 70%, 80%, 2X95%, 3X100%, 3XPropar, 3XParaffin All reagents are 2 hours. Old Protocol with Thermo Scientific Excelsior Processor: 70%, 80%, 2X95%, 3X100%, 3XPropar, 3XParaffin All reagents are 4 hours. Recent Protocol (past year) Thermo Scientific Excelsior Processor: 70% 1hr 85% - 4hrs 95% - 4hrs 2X100% - 4hrs 100% - 5hrs 2XPropar - 4hrs Propar - 5hrs 2XParaffin - 4hrs Paraffin - 5hrs The main questions are: 1) How can we ensure complete fixation? 2) What is causing these brittle/hard areas in our tissues, even for soft tissues? 3) What does over-processing look like in samples? 4) Is our recent protocol okay or does it need adjustments? Thank you in advance for any help or suggestions! Sincerely, Merissa _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet