Nuclear fast red staining

Eyes were enucleated from Fam161a^tm1b/tm1b and WT mice at the age of 1 month and then fixed in 4% paraformaldehyde for 20 min at room temperature. Cornea and lens were removed, eyecup were cryoprotected in a 10–> 20–> 30% sucrose gradient at 4 °C for at least 1 h per each concentration. Eyecup were embedded in optimum cutting temperature medium (Sakura Finetek USA, Torrance, CA, USA) frozen in dry ice and 2-methylbutane slurry, and sectioned at 10 µm onto Superfrost Plus (Thermo Fisher) slides with a cryostat (Leica CM1950, Leica, Wetzlar, Hesse, Germany). Slides were washed with PBS and stained for X-gal with β-Galactosidase Reporter Gene Staining Kit (Sigma-Aldrich, Rehovot, Israel) followed by PBS washing and nuclear fast red staining (Vector Laboratories, Burlingame, CA, USA) according to the protocols supplied by the companies. After an additional PBS wash, slides were coverslipped with Vectamount (Vector Laboratories, Burlingame, CA, USA) and imaged.

Gut tissue was cryo-embedded, sliced and fixed with 4% PFA overnight. Tissue was then incubated in a solution of 3 ml PBS, 1.5 mg nitroblue tetrazozolium (Sigma), 3 mg β-Nicotinamide adenine dinucleotide phosphate (β-NADPH, Sigma) and 1.5 μl Triton X-100 (Sigma) at 37°C for 30 min in the dark (Wallace et al., 2009 (link)). Sections were then washed in PBS and nuclear fast red staining (Vector Laboratories Inc., Burlingame, USA) was performed by incubation with the coloring agent for 20 min at room temperature. The reaction was stopped by washing in PBS. The NADPH-staining was preserved by covering the tissue sections with Kaiser's gelatine (Merck) and cover slips. Samples were visualized and analyzed under morphological peculiarities with Nikon intenselight C-HGFI (Nikon). For quantification, NOS-positive cells in the plexus myentericus region were counted microscopically (25× objective) from 5 non-consecutive longitudinal serial cryosections (16 μm × ~1.5 cm) of each donor (children n = 4, aged donors n = 6).