Ar1068

For alizarin red S staining, the cells were fixed with cold methanol (cat. No. 100230, honey, Ulsan, Korea) for 10 min and washed with deionised (DI) water. Then, cells were stained with 1% alizarin red S (cat. No. A5533, Sigma-Aldrich) solution for 5 min at room temperature (RT). The samples were then rinsed with DI water gently and left to dry.
For von Kossa staining, cells were fixed with 4% (v/v) cold paraformaldehyde (AR1068, BOSTER, Pleasanton, CA, USA) in for 20 min, then rinsed in DI water. Silver nitrate (cat. No. 21572.188, VWR International GmbH, Vienna, Austria) at 5% (w/v) in DI water was added to the fixed cells for 30 min. The cells were exposed to a 100 w UV lamp for 10 min and left to dry. All samples were observed by inverted microscopy (Nikon ECLIPSE Ts2, Nikon, Melville, NY, USA).

Immunofluorescence staining was performed on different days with behavior tests. The procedures were described in previous work [24 (link)]. Briefly, 2 h after the acute drug injection, the mice were anesthetized with tribromoethanol (20 mg/kg), perfused with phosphate-buffered saline (PBS), and 4% paraformaldehyde (AR1068; Boster Biological Technology, Wuhan, China) for pre-fixation. The brain tissue was removed, placed in 4% paraformaldehyde, and stored at 4 °C for 24 h, then dehydrated with 20% and 30% sucrose solutions for 2 days. The OTC-embedded tissue was cut into 40-μm sections using a freezing microtome (CM1950; Leica, Wetzlar, Germany). After permeabilization and blocking, the sections were incubated with primary anti-c-fos antibody (1:500, rabbit, #2250; Cell Signaling Technology, Danvers, USA) at 4 °C for 20 h and then washed three times with PBS. Afterward, the sections were incubated with the secondary Alexa Fluor488-conjugated donkey anti-rabbit antibody (1:500, A21208; Invitrogen, Carlsbad, USA) at room temperature for about 2 h and washed with PBS. To stain the nuclei, 4′,6′-diamidino-2-phenylindole (DAPI, 0.1 μl/ml) was used for about 5 min. Sections were washed with PBS and covered on a glass slide. Brain slices were imaged using a confocal microscope (LSM 880 with Airyscan; Zeiss, Jena, Germany).

Eyes were enucleated and fixed in 4% PFA in PBS (AR1068; Boster) for 1 hour at room temperature. Retina and RPE/choroid were dissected with fine forceps and four radial cuts were made to flatten the retina. Tissues were blocked and permeabilized while slowly shaking at room temperature for 1 hour in 1 mL blocking buffer (0.5% Triton X-100 and 1% skimmed milk in PBS). Primary antibodies (see Supplementary Table S1) were diluted in blocking buffer and tissues were incubated in primary antibodies at 4°C overnight. After 3 washes with PBS, matching secondary antibodies labeled with a fluorophore were incubated at 4°C overnight. Nuclei were stained with 1:1000 diluted DAPI solution (62248; ThermoFisher). Tissues were washed three times in PBS. SecureSeal Imaging Spacer (SS8 × 9; Biotrend) were applied to glass slides and single retinae were transferred into each well of the spacer. Then, 8 µL Vectashield (Vectorlabs) was added to each tissue and sealed with a cover slip. Tissues were imaged with a laser-scanning microscope (LSM710; Zeiss) or an Opera Phenix High-Content Screening System (PerkinElmer).

Cells grown on glass cover slips were washed six times with DPBS, fixed with 4% (w/v) formaldehyde in PBS (BosterBio #AR1068) in DPBS for 15 min in the dark at room temperature (RT). Thereafter, cells were washed again three times with DPBS, permeabilized with permeabilization buffer containing 0.02% (w/v) saponin (Sigma-Aldrich #47036) and 1% (w/v) bovine serum albumin (Sigma-Aldrich #A6003) in DPBS at RT. Cells were incubated with blocking buffer containing 1% (w/v) bovine serum albumin in DPBS at RT, then incubated with primary antibody (1:100 dilution in blocking buffer) for 2 h at RT, washed three times with DPBS and incubated with secondary antibody (1:1000 dilution in blocking buffer) for 45 mins at RT. Cover slips were washed three times with DPBS and mounted on glass slides with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich #F4680). Slides were dried at RT in the dark overnight before imaging. Information on the antibodies used is available in the reagent sheet.

Eyes were enucleated and fixed in 4% paraformaldehyde (AR1068; Boster Bio, Pleasanton, CA, USA) for 48 hours at 4°C. Whole eyeballs were dehydrated and infiltrated with paraffin using a tissue processor (Tissue-Tek VIP 6; Sakura Finetek USA, Inc., Torrance, CA, USA) and subsequently embedded into paraffin blocks on a HistoCore Arcadia Embedding Center (Leica Biosystems, Melbourne, Australia). Three-micrometer sections were cut at the level of the optical nerve and hematoxylin and eosin and Masson's Trichrome staining was performed according to standard protocols. Immunofluorescence staining was carried out on the automated Leica Bond platform (Leica Biosystems) using the Opal Multiplex IHC Kit (Akoya Biosciences, Menlo Park, CA, USA). Antibodies used in this study and details about the antigen retrieval are listed in the Table. Nuclei were stained with spectral DAPI (FP1490; Akoya Biosciences) and slides mounted with ProLong Antifade Mounting Medium (P36961; Invitrogen, Carlsbad, CA, USA). Tissues were imaged using an Axio Scan.Z1 slide scanner (magnification ×20; Carl Zeiss Microscopy GmbH, Jena, Germany). Iba1⁺ and F4/80⁺ area was quantified using HALO image analysis software (Indica Labs, Albuquerque, NM, USA).