Axioscan 7 slide scanner

Freshly dissected mouse bones were fixed with 4% paraformaldehyde (PFA) (15,710 S, Electron Microscopy Sciences) for 4 hr at 4 °C. After samples were washed with PBS, decalcification was processed with 0.5 M EDTA for 5 days. Samples were incubated with infiltration solution (20% sucrose (S7903, Sigma) plus 2% polyvinylpyrrolidone (Sigma, 9003-39-8) in PBS) until they sank to the bottom of the tube. Embedding was performed with OCT (Sakura Finetek,4583) and samples were stored at −80 °C. The samples were sectioned at 10 μm in thickness using a Leica cryostat. Frozen sections were thawed at room temperature and rehydrated with PBS, permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature, and blocked for 1 hr with 5% BSA in PBS (blocking buffer). Primary antibodies were freshly diluted in blocking buffer and were incubated with the slices overnight at 4 °C. After washing three times with PBS, secondary antibodies (1:2000 dilution with blocking buffer) were incubated for 1 hr at room temperature. Samples were then washed and mounted with antifade mounting solution with DAPI (Life technologies, P36941). Imaging was employed using a Zeiss Axioscan 7 Slide Scanner.

Dewaxed and rehydrated tissue sections were stained for 1 h with Sirius Red F3B (Direct Red 80, #365548, Sigma-Aldrich, Saint Quentin-Fallavier, France) solution at 1 g/L in a saturated aqueous solution of picric acid (1.3%). After two quick washes in 0.5% acidified water, the sections were dehydrated, mounted, and scanned using the Axioscan 7 slide scanner (Zeiss) under brightfield filters.

Wholemount preparations of circular muscle, myenteric plexus and longitudinal muscle were prepared by removing the mucosa and submucosa from the fixed tissue. Preparations were blocked in NHS (10% normal horse serum in PBS with 1% Triton X-100) for 30 min at room temperature (RT) and then incubated with antibodies against markers of different classes of colon innervating neurons (Table S1) overnight at 4°C. The wholemounts were then washed (three times for 10 min) in PBS before incubation with secondary antibodies (Table S2) for 1 h at RT. Preparations were given three subsequent 10 min washes in PBS and then mounted on glass slides using mounting medium (S3023 non-fluorescent mounting medium, Dako Corporation, Carpinteria, CA, USA). An unstretched 1-week-old KO colon and a 13-day-old WT colon were stained whole without further dissection. Antibody incubations were increased to four nights at 4°C for the primary antibody and one night at RT for the secondary antibody to allow adequate antibody penetration into these samples. The entire colon was imaged as a z-stack tile scan on an Axioscan 7 Slide Scanner (Zeiss, Sydney, Australia).