Cryochrome

Nissl staining was used to recognize the morphology of neural cells and performed as follows: the tissue was dehydrated in a graded alcohol series of increasing concentration, then treated with distilled water, cresyl violet solution, distilled water, a graded alcohol series of increasing concentration, and xylene.
The procedure for cresyl violet (CAS 10510-54-0; Sigma-Aldrich) staining was similar to that described previously [27 (link),28 ]. Tissue of the right vagal plexus (RVP) structure localized between the connection of the RVN to the stomach and celiac plexus was removed and further postfixed with Carnoy fixative for 24 h. VP was treated with a graded sucrose series of increasing concentration with changes every 24 h. After this, the RVPs were cut into 10 µm sections with a cryostat (Thermo Scientific, Waltham, MA, USA. 77210163) with a resin of inclusion (Thermo Scientific Shandon Cryochrome, Waltham, MA, USA) at −25 °C.

Whole retinal explants were fixed at room temperature in 4% paraformaldehyde (PFA) for 0.5 h. Following washes, retinas were cryo-protected in 15% sucrose in 1xPBS for 1 h, 20% sucrose for 1 h and 30% sucrose overnight, all at 4 °C. Retinas were submerged and frozen in cryochrome (Thermo Scientific, Waltham, US) and sectioned on a cryostat (Leica, Wetzlar, Germany). Sections (7 μm for TUNEL and 20 μm for IF on microglia) were collected on Superfrost glass slides (Fisher Scientific, Waltham, US) and stored at −80 °C. Sections were blocked and permeabilized with 0.1% Triton X and 5% donkey serum in 1xPBS for 30 min and incubated with primary antibody diluted in 5% donkey serum overnight at 4 °C. Table 1 lists the details of all primary antibodies used. Following washes, sections were incubated with secondary antibody (Alexa Fluor donkey anti-rabbit/rat with either a 488 or 594 fluorescent probe; Molecular Probes &) and Hoechst 33342 nuclear stain (1:10,000; ThermoFischer) for 1 h at room temperature. Eliminating the primary antibody in solution served as a negative control (Supplementary Figure S1). Sections were mounted using Mowiol (Sigma) with Dabco anti-fade agent (Sigma).

Eyes were enucleated and fixed at room temperature in 4% paraformaldehyde (PFA) for 1.5 h. Following washes, eyes were cryo-protected in 15% sucrose in 1xPBS for 1 h, 20% sucrose for 1.5 h and 30% sucrose overnight, all at 4°C. Eyes were submerged and frozen in cryochrome (Thermo Scientific, Waltham, US) and sectioned on a cryostat (Leica, Wetzlar, Germany). Sections (7μm) were collected on Superfrost glass slides (Fisher Scientific, Waltham, US) and stored at -80°C. Sections were blocked and permeabilized with 0.1% Triton X and 5% donkey serum in 1xPBS for 30 min and incubated with primary antibody diluted in 5% donkey serum overnight at 4°C. Table 1 lists the details of all primary antibodies used. Following washes, sections were incubated with secondary antibody (Alexa Fluor donkey anti-mouse/rabbit/goat with either a 488 or 594 fluorescent probe; Molecular Probes &/or FITC–PNA (Vector Labs, Burlingame, US.)) with the nuclear stain, Hoechst (Thermo Scientific) for 1 h at room temperature. Eliminating the primary antibody in solution served as a negative control. Sections were mounted using Mowiol (Sigma) with anti-fade agent Dabco (Sigma). Images were taken from central and peripheral regions of the retina. Regions identified as central and peripheral are shown in S1 Fig.