Research Specialist II Boston University Chobanian & Avedisian School of Medicine Boston, Massachusetts, United States
Abstract Body : For optimal immunohistochemistry (IHC), tissue needs to be treated with fixative (to preserve proteins) and cryoprotected (to avoid freezing artifacts) before it can be cut into slices on a cryostat or on a freezing microtome. In many laboratories, tissue is collected fresh and flash-frozen for molecular studies after long-term storage. Development of a method that allows the same slab of fresh-frozen tissue to be used for both molecular and histological examination maximizes use of precious harvested tissue and makes the most of a laboratory’s frozen tissue archive. Here, we present a novel, reliable, and inexpensive method of thawing fresh-frozen tissue in such a way that preserves it for IHC labeling and is simple to execute. First, glycerol was diluted by 2% DMSO to 30%, 20%, and 10% concentrations. The 30% and 20% glycerol solutions were chilled to the point of near-freezing (-9°C and -5°C, respectively). Tissue was first submerged in the chilled (-9°C) 30% glycerol solution for 5 minutes, then transferred to the chilled (-5°C) 20% glycerol solution for 6 hours. Finally, tissue was transferred to the 10% glycerol solution overnight and kept at 4°C for an additional 24 hours. Following the step-down glycerol thawing, tissue was fixed by immersion in a 4% paraformaldehyde solution for 2 days. Finally, the tissue was cut on a vibratome into 40µm-thick sections and subjected to an IHC labeling protocol to label neurons, glial cells, and vascular structures. The IHC labeling protocol used on this thawed tissue was identical to that used on tissue prepared specifically for that purpose. Additionally, quantitative birefringence microscopy (qBRM) was used to assess myelin integrity. The results are compared to tissue that was perfusion fixed and cryopreserved for optimal IHC.
