Tissue tek oct compound

Mice were anesthetized by isoflurane inhalation, and the distal aorta was cut to exsanguinate the animal. The mice were perfused via the right ventricle with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in PBS. The lungs were then separated into lobes and immersed in PBS containing 10% sucrose at 4 °C. The sucrose density was consecutively changed to 16, 18, and 20%. The lungs were then rinsed with PBS containing 50% Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) and frozen in Tissue-Tek OCT compound at − 80 °C. Frozen sections were cut using a cryostat (to a thickness of 7 μm) at − 20 °C.

After the animal was removed from the stereotaxic frame, still under anesthesia, the femoral vein was exposed and a catheter inserted. Blood was withdrawn into heparinized micro-centrifuge tubes, centrifuged at 400×g for 8 min, and the aspirated plasma layer stored at −80 °C for later analysis. The cell pellet was resuspended in red blood cell lysis buffer (Biolegend, California, USA), incubated on ice for 5 min, centrifuged (400×g, 8 min), and frozen. The animals were perfused with 100 ml ice-cold PBS, pH = 7.2, and decapitated under deep isoflurane anesthesia.
One random brain hemisphere was frozen in isopentane and stored at −80 °C and the other processed for mitochondrial isolation (see below).
Pieces of tissue from the cervical, thoracic, and lumbar/sacral regions of the spinal cord were frozen in isopentane and stored at −80 °C, processed for mitochondrial isolation, or frozen in isopentane at −80 °C, and embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek, Alphen Aan Den Rijn, The Netherlands) for cutting. The right liver lobe was frozen in isopentane and stored at −80 °C. The optic nerve was frozen in isopentane at −80 °C and embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek) for sectioning.