Dapi mounting medium

Corneas of mice were dissected under a binocular microscope and fixed with 4% PFA (P0099; Beyotime) for 1 hour. Then, mouse corneas were permeabilized with 0.1% Triton X-100 (P0096; Beyotime) in PBS for 5 minutes and blocked in 3% bovine serum albumin (BSA) in PBS for 30 minutes. Subsequently, anti-zonula occludens-1 (ZO-1) antibody (40-2200; Invitrogen) and anti-sodium potassium ATPase (Na⁺-K⁺-ATPase) antibody (Ab76020; Abcam) in 1% BSA-PBS was used to incubate the corneal cups at 4°C overnight, respectively. After that, secondary anti-rabbit fluorescein isothiocyanate (FITC; ab150077; Abcam) was applied to conduct the secondary antibody incubation. Corneal cups were flattened by four radial cuts and mounted by DAPI mounting medium (0100-20; Southern Biotech). The graphs of ZO-1 and Na⁺-K⁺-ATPase staining were captured using the laser confocal scanning microscope (Leica SP8; Wetzlar, Germany).
For periodic acid–Schiff (PAS) staining, paraffin tissue slices of mouse corneas were dewaxed and oxidized by 0.5% periodate acid solution. Then, corneal slices were stained in PAS reagent in the dark for 30 minutes. Subsequently, nuclear dying using hematoxylin, dehydration, and slice seal were performed. The images of PAS staining were obtained using an optical microscope.

30 μm transverse (immunofluorescent) or 20 μm longitudinal (in situ) cryosections were cut using a CM1950 cryostat (Leica Biosystems). For ChAT and NeuN immunostaining antigen retrieval was performed prior to permeabilization and blocking by boiling sections in 10 mM sodium citrate buffer, pH 6.0 for 3 min. Sections were permeabilized and blocked in PBS containing 0.2% Triton-X, 1% BSA, 10% donkey serum (Jackson ImmunoResearch), and M.O.M blocking reagent (Vector Laboratories) for 1 hour at room temperature. Slides were then incubated with primary antibodies [(HA (1:100, #3724, Cell Signaling), ChAT (1:100, #AB144P, Millipore), NeuN (1:100, #MAB377, Millipore), CD11b (1:100, #MCA711, Biolegend), CD68 (1:100, #MCA1957, Biolegend), Synapsin (1:250, #5297S, Cell Signaling), Tuj1 (1:500, #T8578, Sigma-Aldrich)], MCP3 (CCL7, 1:100, #ab228979, Abcam) in PBS containing 0.2% Triton-X, 1% BSA overnight at 4°C (for most antibodies) or at room temperature (for ChAT and NeuN staining). The following day sections were washed in PBS and stained with the fluorescently conjugated α-bungarotoxin (for NMJ analysis) and/or appropriate secondary antibodies (Biotium) in PBS containing 0.2% Triton-X for 1 hour at room temperature. After PBS washes, slides were cover slipped with DAPI mounting medium (Southern Biotech) and imaged on a confocal microscope (Leica SP5).