Positively charged glass slides

Samples were held at −20°C for 24 h to bring samples to an appropriate temperature for cryosectioning. After being embedded in frozen section compound (VWR International, Westchester, PA), 5-μm thick cryosections were collected from the intestinal samples, transverse to the length of the intestine, using a Leica CM 1950 cryomicrotome. The cryosections were mounted on positively charged glass slides (VWR International; 5 cryosections per slide) and stored at 4°C for no more than 48 h before immunofluorescence staining.

Rats were euthanized by transcardial perfusion. Rats were given a lethal dose of ketamine and dexmedetomidine (as above), and their thorax was opened to expose the heart. The perfusion cannula was inserted through the left ventricle into the aorta and a small incision was made in the right atrium. The rats were perfused with 300 mL phosphate-buffered saline (PBS) until the blood was cleared, followed by 400 mL of 4% paraformaldehyde (PFA) in PBS (0.1 M, pH 7.4) until tissue fixation was achieved. The spinal cord was dissected out, kept in 4% PFA for 24 h at 4 °C, and then transferred to 30% sucrose in PBS (0.1 M, pH 7.4) for at least 48 h. Then, a 12 mm long segment centered at the injury epicenter was cut out, embedded in NEG-50 medium (ThermoFisher Scientific, Waltham, MA, USA), and frozen at –20 °C for approximately 2 h. The tissue was longitudinally sectioned on a Leica cryostat at 20 µm thickness. Sections were mounted in series on positively charged glass slides (VWR, Radnor, PA, USA) and stored at –20 °C until further use.