Oct tissue freezing medium

The intact C57BL/6 male mice were deeply anaesthetised by intraperitoneal urethane injection (2.4 g/kg) and transcardially perfused with 20 mL of ice-cold 0.1 M phosphate-buffered saline (PBS, pH: 7.4) followed by 150 mL 4% paraformaldehyde (PFA) solution in Millonig buffer (pH 7.4) for 15 min. After perfusion, the brains with OBs were removed, and collected into PFA for 36 h for postfixation at 4 °C. Olfactory epithelia were also removed and collected into 4% PFA containing 30% sucrose for 36 h postfixation, to ensure cryoprotection. The brains with the OBs were coronally sectioned using a Leica VT1000 S vibratome (Leica Biosystems, Wetzlar, Germany). Five series of 30 µm sections were gathered and stored in antifreeze solution (20% ethylene glycol, 30% glycerol and 0.1 M sodium-phosphate buffer) at −20 °C. OE samples were embedded in OCT tissue freezing medium (Leica), and five series of 17 µm transversal sections were obtained using a cryostat (Leica CM1950, Nussloch, Germany). Then, sections were taken on SuperFrost Ultra Plus adhesion slides (Thermo Fisher Scientific, Braunschweig, Germany, Cat. No.: 10417002), and stored at −20 °C.

The permeability of the blood-testis barrier was assessed using Inulin-FITC (Sigma, F3272). Briefly, a total of 20 μL Inulin-FITC (10 mg/mL in PBS) was injected into the interstitium of the testes. The animals were euthanized 40 min later, and the testes were immediately collected and embedded in optimal cutting temperature (OCT) tissue freezing medium (Leica). Nuclei were stained with DAPI, and fluorescence images of the cryosections were captured with a fluorescence microscope (Olympus).

Lucifer yellow dye (1 mg/mL; Sigma-Aldrich, USA) was topically applied on the epidermis for 1 h. Excess Lucifer yellow was washed away with 1 × PBS, and the skin tissues were cryofrozen with OCT tissue freezing medium (Leica, USA). Tissues were cryosectioned at 8 μm and mounted with Hoechst 33,342 dye (Life Technologies, USA). Images were taken using the LSM 710 confocal microscope with the Zeiss EC Plan-NEOFLUAR 20×/0.5 NA objective. Analyses were performed with ZEN 2012 Light Edition software (Carl Zeiss, Germany).

Adult (2–3 months) BALB/c mice obtained from our in-house breeding colony were used in all experiments. ICV cannulation was performed under aseptic conditions as described previously^{58 (link)}. In brief, 4 μg/μL polybrene was incubated at 37 °C for 15 min, and ICV injection of 5 μL mixture was performed for ≥20 min.
At day 3 after ICV injection, Motor Performance Test-Accelerating Rotating Rod Test was performed. The rotating rod apparatus was used to evaluate motor function. The mice were placed on the rod (3 cm in diameter) for four trials. Each trial lasted for a maximum of 5 min, during which the rotating rod underwent linear acceleration from 0 to 30 rpm and then remained at maximum speed. Animals were scored for their latency to fall (in seconds) in each trial.
Then the mice were deeply anesthetized and euthanized by transcardiac perfusion with fixative solution (4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, 50 mL/animal). The brains were harvested and postfixed in 4% paraformaldehyde, dehydrated in increasing concentrations of sucrose (30–60%), infiltrated in OCT Tissue Freezing Medium (380148, Leica), and plastic-embedded. Frozen sections (15 μm) were cut, mounted on coverslips, and then dried at room temperature and stained with NeuN (ab128886, Abcam, 1:500) and GFAP (Z033429, Dako, 1:1000).