Enhanced immunostaining permeabilization buffer

Cu B (Lot#23114, 99.91% pure) was derived from MedChemExpress (Shanghai, China). The Cell Counting Kit-8 (CCK-8), 4% paraformaldehyde fix solution, enhanced immunostaining permeabilization buffer, DAB horseradish peroxidase color development kit, QuickBlock™ blocking buffer for immunol staining, and QuickBlock™ primary and secondary antibody dilution buffers for immunohistochemistry were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/PI kits were from Yeasen (Shanghai, China).

The cells were fixed with precooled 4% paraformaldehyde solution for 30 minutes, washed once with PBS, lysed for 10 min with Enhanced Immunostaining Permeabilization Buffer, washed twice with PBS, and then 5 μl of terminal deoxyribonucleotidyl transferase (TDT) enzyme and 45 μl of FITC labeling solution were added, and covered with antievaporation membrane followed by incubation at 37°C for 1 h in the dark; then, the cells were washed three times with PBS. Next, 200 μl of Antifade Mounting Medium with DAPI was added to every well, and the number of apoptotic cells in each group was counted by fluorescence microscopy. Excited by blue-green light, green apoptotic nuclei can be observed, and excited by ultraviolet light, all nuclei are blue. The ratio of green nuclei to blue nuclei is the apoptosis rate. The average apoptosis rate was obtained by observing five fields from each group. One Step TUNEL Apoptosis Assay Kit, 4% Paraformaldehyde Fix Solution, Enhanced Immunostaining Permeabilization Buffer, and Antifade Mounting Medium with DAPI were purchased from Beyotime (Shanghai, China).

The model rats were euthanized at 7 days after the intravitreal injection of liposomes. Immediately after the eyeball was removed, it was fixed in 4% paraformaldehyde for 2 h at 4°C, dehydrated overnight with 30% sucrose solution, embedded in an optimal cutting temperature compound (OCT) embedding bottom box (17 × 17 × 5 mm), and stored at −80°C. Slices with thicknesses of 8 μm were created using a frozen slicer at −22°C. The slices were placed on adhesive slides and dried at room temperature for more than 30 min before proceeding to the next step. For immunofluorescent staining, the retinal sections were permeabilized with enhanced immunostaining permeabilization buffer (Beyotime) for 20 min, blocked with blocking buffer (Beyotime), and incubated overnight at 4°C with primary antibodies: Class III β‐TUBULIN (Tuj1 mAb) (Beyotime), Rabbit anti‐IBA1/AIF‐1, rabbit anti‐FOXO3A and rabbit anti‐SIRT1(Cell Signaling Technology). After rinsing three times with PBS, the sections were incubated with secondary antibodies at room temperature for 2 h, rinsed three times with PBS, and counterstained with DAPI for 5 min. The slides were then sealed with cover glass for observation by CLSM.

Cell proliferation was measured using a 5-ethynyl-2’-deoxyuridine (EdU) Cell Proliferation Assay Kit (C0075S, Beyotime, Shanghai, China) as described in the manufacturer’s instructions. CRC cells were cultured at a density of 3 × 10⁵ cells/well in a 12-well plate. After attachment, the cells were exposed to EdU labeling medium for approximately 2 hours at 37°C. Afterwards, the cells were fixed with 4% formaldehyde for 15 minutes and treated with enhanced immunostaining permeabilization buffer (P0097, Beyotime, Shanghai, China) for 15 minutes at room temperature for permeabilization, after which the click reaction mixture were added to the cells and incubated for 30 minutes. Next, the nuclei of the cells were stained with Hoechst 33342 in the dark at room temperature for 10 minutes and visualized under a confocal laser scanning microscope (ZEISS, Jena, Germany). Experiments were performed in triplicate.

Cell apoptosis in CA1 and CA3 areas of rat hippocampus from Control, VB₆ deficiency, and VB₆ supplement groups was determined using Colorimetric TUNEL Apoptosis Assay Kit (C1091, Beyotime, China). The frozen hippocampal sections were treated with Immunol Staining Fix Solution (P0098, Beyotime, China) at room temperature for 30 minutes, followed by the rinse using PBS twice. Then, each section was immersed into Enhanced Immunostaining Permeabilization Buffer (P0097, Beyotime, China) for 5 minutes, blocked with 0.3% H₂O₂ in PBS for 20 minutes, and reacted with 50 μl prepared biotin-labeled solution at 37°C in a wet box for 1 hour without light. After the reaction was terminated, the sections were subjected to color development, including the treatment of both streptavidin–horseradish peroxidase (HRP) (50 μl) and 3,3′-diaminobenzidine (DAB, 0.2 ml) according to the manufacturer's specification. Following dehydration with gradient alcohol and hyalinization with xylene, the number of positively apoptotic cells in the hippocampal CA1 and CA3 by cells/0.01 mm² was counted using a light microscope (×100 magnification, CX23, Olympus, Japan). Three hippocampal hemispheres from six consecutive serial sections were used.

The RIR‐injured rats were euthanized at 7 days after the intravitreal injection of Rh‐GFFYE. Immediately after removal, the eyeball was fixed in 4% paraformaldehyde for 2 h at 4 °C, dehydrated overnight in 30% sucrose solution, embedded in an optimal cutting temperature compound embedding bottom box (17 × 17 × 5 mm), and stored at −80 °C. Slices with thicknesses of 8 µm were created using a frozen slicer at −22 °C. The slices were placed on adhesive slides and dried at room temperature for >30 min before proceeding to the next step. For immunofluorescent staining, the retinal sections were permeabilized with enhanced immunostaining permeabilization buffer (Beyotime, Beijing, China) for 20 min, blocked with blocking buffer (Beyotime, Beijing, China), and incubated overnight at 4 °C with the following primary antibodies: anti‐β‐III‐TUBULIN, anti‐IBA1, anti‐GFAP, anti‐BRN3A (Abcam, UK), anti‐CD86, and anti‐CD206 (Cell Signaling Technology, Danvers, MA, USA). After rinsing three times with PBS, the sections were incubated with secondary antibodies at room temperature for 2 h, rinsed three times with PBS, and counterstained with DAPI. Sealed with a cover glass, the slides were then observed by CLSM.