R0156

Sections for immunohistochemistry were fixed in 2% PFA, washed three times in PBS, and blocked in swine normal serum (1:20) for 15 min. Sections were then incubated with rabbit primary antibodies (type I collagen, 1:80, Abcam, ab34710; type III collagen, 1:100; Abcam, ab7778; and NK-1R, 1:50, Santa Cruz, SC-15323) for 60 min at 37 °C. Washing and blocking was repeated before the sections were incubated with TRITC-conjugated swine-anti-rabbit secondary antibodies (1:40, Dako, R0156) for 30 min at 37 °C. After a final washing step the slides were mounted in medium containing DAPI (Vector Laboratories, code: H-1200). Slides were examined in a Zeiss Axioskop 2 plus microscope equipped with an Olympus DP70 camera.

Cells were plated onto glass coverslips and allowed to attach. Cells were fixed with 4% paraformaldehyde (AlfaAesar^®) for 15 min. Where necessary, prior to fixation, soluble protein was pre-extracted using 0.5% Triton X-100 in KK2 for 5 min at 4 °C. Cells were then permeabilised in 0.5% Triton X-100 in KK2 for 10 min, and blocked in 1% bovine serum albumin (Sigma^®) for 1 h. Coverslips were stained with primary antibody (2 h, room temperature) against H3S10-P (1:500, Bethyl^® A301-844A-T) or γH2AX (1/500, Abcam^® ab11174), washed extensively in KK2-0.01% Tween-20^®, and stained with fluorescently labelled secondary antibody (2 h, room temperature) anti-mouse FITC (1:500, DAKO^®, F0232) or anti-rabbit TRITC (1:500, DAKO^®, R0156). Following further washing, cells were mounted onto slides in Vectashield^® containing DAPI (Vector Laboratories). Samples were visualised using a microscope Zeiss IX71 equipped with a 10X dry objective and a 100X oil immersion objective lens and a Hamamatsu^® Orca-R² camera. Pictures were analysed with ImageJ^® software.

The enucleated eyes were routinely fixed in 10% buffered formalin and embedded in paraffin. The paraffin-embedded sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. One set of sections was stained with H &E for light microscopy examination. The remaining sets were used for immunofluorescence studies. The sections were first blocked with 2% BSA in PBST, and the primary antibodies, anti-SYPH (1∶1000 dilution, 17785-1-AP, Proteintech, China) and anti-SYT1 (1∶50 dilution, 14511-1-AP, Proteintech) were applied on the sections at 4°C overnight. After washing three times with PBST, the slides were then incubated with the secondary antibody (1∶200 dilution, #R0156, Dako, Denmark) for 1 h. The sections were covered with DAPI and anti-fading medium after extensive washing with PBS and imaged via an Olympus FV1000 Confocal Laser Scanning Microscope (Olympus, Japan).