Surgipath fsc 22 clear frozen section compound

Pimonidazole (Hypoxyprobe, Inc., Burlington, MA, USA) was injected intraperitoneally at the dose of 60 mg/kg body weight. The mice were sacrificed 1 h after the injection, and the tumor tissue was flash-frozen in Surgipath FSC 22 clear frozen section compound (Leica Biosystems Richmond, Inc., Richmond, IL, USA). The tissue was cryosectioned at a thickness of 7 μm and fixed in ice-cold acetone (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The sections were incubated with FITC-Mab1 (clone 4.3.11.3; Hypoxyprobe, Inc., Burlington, MA, USA) overnight at 4 °C to stain for Pimonidazole [39 (link)]. The slides were then mounted in Fluoroshield mounting medium with DAPI (Sigma-Aldrich, St. Louis, MO, USA) and stored at 4 °C. Images of three random fields of view (FOVs) were acquired for each sample using an upright fluorescence microscope (DM2500; Leica Microsystems, Wetzlar, Germany) and a color CCD (DP73; Olympus, MA, USA). ImageJ software was used to quantify the mean fluorescence intensity (MFI) [39 (link)], and statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test in GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).

After electrophysiological evaluation, all rats were anesthetized, and the extensor hallucis longus muscle was harvested and perfused with 50 mL of 0.9% saline and then 200 mL of 4% PFA in 0.1 M phosphate buffer. The tibialis anterior muscle was then harvested and weighed immediately. The peroneal nerve was harvested and divided into proximal and distal halves. The proximal peroneal nerve, including the cell transplant site, was cryoprotected with sucrose and frozen with isopentane cooled with liquid nitrogen into suitable tissue forms containing compound (Surgipath FSC 22 Clear Frozen Section Compound, Leica Biosystems, Nussloch, Germany). The distal half of the peroneal nerve was fixed with 2% glutaraldehyde/4% PFA in phosphate buffer and embedded in the Epon.