Tissue tek viptm processing machine

Mist1^CreERT2;R26^tdTomato male mice (6–8 weeks, n=3) were given one dose of tamoxifen (0.25 mg/g body weight) (MP Biomedicals) by gavage 3 days before duct ligation. On the day of surgery, mice were pre-emptively given Buprenorphine SR (1 mg/kg, Wildlife Pharmaceuticals) subcutaneously as analgesia, and then anesthetized with an intraperitoneal injection of ketamine HCl (80 mg/kg, JPH Pharmaceuticals) and xylazine (8 mg/kg, Lloyd Laboratories) in saline. A ventral incision was made on left side of the neck to isolate the main secretory duct of the left SMG. The SMG main duct was ligated using a titanium hemostatic clip (#R9180, Vitalitec Int.). The right contralateral gland served as the sham-operated control. After 14 days, mice were re-anesthetized, and the clip was removed. Following the de-ligation, the SMGs were either immediately isolated or allowed to regenerate for 14 days. Tissue was isolated and fixed in 4% paraformaldehyde at 4°C overnight. The tissue was processed using a Tissue-Tek VIPTM processing machine (Sakura Finetek USA, Inc.) before paraffin embedding. Sections (5 μm) were cut using a Leica RM2125 rotary microtome and collected on Superfrost Plus slides (Thermo Fisher Scientific).

Mist1^CreERT2;R26^tdTomato mice were administered tamoxifen (0.25 mg/g body weight) by gavage daily for 4 days (Aure, et al., 2015b (link)). Mice (n = 3) were sacrificed for gland removal 3 days after the last tamoxifen gavage. Mist1^CreERT2;R26^tdTomato mice not treated with tamoxifen served as controls to detect non-specific recombination. SMG were fixed overnight in 4% PFA. For frozen sections, whole glands were transferred through a sucrose gradient (5%, 10%, 15% sucrose for 30 minutes each) and incubated in 30% sucrose : OCT (50:50) solution overnight. Samples were embedded in OCT and sectioned at 10 μm using a cryostat. For paraffin sections, SMG was processed in a Tissue-Tek VIPTM processing machine (Sakura Finetek USA, Inc.) and embedded in paraffin. Sections (5 μm) were cut using a Leica RM2125 rotary microtome.