Ultramount aqueous permanent mounting medium

Cells were transfected with either WT or variant METTL23-FLAG vectors using ViaFect transfection reagent (Promega) on 24-well coverslips (MS-92132, Sumitomo Bakelite) in 24-well plates. After fixation with 4% (PFA) and permeabilization with 0.3% Triton X-100 in PBS for 10 minutes, the transfected cells were blocked with protein block serum-free (Dako) for 1 hour and incubated with the primary antibody, anti-METTL23 antibody (1:500; Thermo Fisher Scientific; PA5-71814), and anti-FLAG antibody (1:500; CST; 9A3) overnight at 4°C. Then, Alexa Fluor 568–conjugated goat anti–mouse IgG antibodies (1:500; Invitrogen, Thermo Fisher Scientific), Alexa Fluor 488–conjugated goat anti–rabbit IgG antibodies (1:500; Invitrogen, Thermo Fisher Scientific), and DAPI (1:500; Dojindo) were used to detect FLAG, METTL23, and DNA signals, respectively. The cells were mounted with Ultramount Aqueous Permanent Mounting Medium (DakoCytomation) and visualized under a confocal fluorescence microscope (LSM700; Zeiss).

Eyes were dissected and immunostained in flat mounts as described previously (20 (link)). Briefly, dissected eyes were fixed in 4% PFA overnight at 4°C and carefully dissected. Nonspecific binding was prevented by blocking with Dako’s serum-free blocking buffer, and all specimens were incubated with anti-Brn3a antibody (1:200; AB5945, MilliporeSigma) at 4°C for 5 nights. After incubation with Alexa Fluor 568–conjugated rabbit anti–rabbit IgG (1:1,000; Life Technologies, Thermo Fisher Scientific), retinas were mounted in Ultramount Aqueous Permanent Mounting Medium (DakoCytomation). Four micrographs (700 × 700 μm) per single retinal specimen from the mid-peripheral region of quadrants 1.5–2.0 mm from the optic head were imaged using an LSM 700 confocal fluorescence microscope (Zeiss). Brn3a⁺ RGCs were counted using ImageJ (NIH) and averaged for at least 3 retinas per group.

Surgical specimens from patients undergoing radical prostatectomy were fixed in 10% buffered formalin, embedded in paraffin, and then cut into 4 µm thick serial sections. The first sections were stained with hematoxylin and eosin and used for pathological diagnosis of the inflamed region. The remaining three sections were subjected to immunohistochemical analyses, as described previously [23] (link). Briefly, deparaffinized sections were autoclaved in 0.1 M citrate buffer, blocked with bovine serum albumin, and then incubated with primary antibodies in PBS containing 2% bovine serum albumin. Secondary antibody incubations and signal enhancement reactions were performed using the Histofine Simple Stain kit (Nichirei) and the color was developed using aminoethlcarbazole (Impact AEC; Vector Laboratories). The sections were counterstained with hematoxylin for nuclear visualization and then mounted using Ultramount Aqueous Permanent Mounting Medium (Dako). Images were recorded using a microscope (BX51; Olympus) equipped with a CCD camera (DP72; Olympus).