Vectashield vibrance antifade mounting medium with 4 6 diamidino 2 phenylindole dapi

The frozen sections were stained using antibodies described in Table 1. Before staining, the sections were fixed in acetone (−20°C) for 10 min, washed in phosphate buffered saline (PBS), and blocked using 12% bovine serum albumin (BSA) in PBS for 60 min at room temperature. After washing in PBS, the sections were incubated sequentially with antibodies at room temperature for 60 min, followed by washing 3× with PBS after staining. Sections were then mounted using Vectashield Vibrance Antifade Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI, Vector Laboratories).
Images of the stained sections were acquired with a confocal laser scanning microscope (Zeiss LSM 800), using a Plan‐Apochromat 20×/0.8. All samples were imaged within 12 h after being mounted, and imaged using identical settings optimized to obtain maximum signal with minimum background. AF‐488 was excited at 488, and fluorescence was detected at 515–620 nm. AF‐647 was excited at 640 nm while emission was detected at 656–700 nm. DAPI was excited at 405 nm while detecting fluorescence at 400–515 nm.

PC12 cells were seeded onto poly-L-lysine coated coverslips and cultured in normal serum medium for 24 h. On the following day, the culture medium was replaced with low-serum RPMI medium, and the cells were treated with corticosterone (300 µM) in the absence or presence of fisetin (40 μM) for 24 h. After incubation, the coverslips were carefully lifted and placed onto slides. Subsequently, they were mounted using 20 µL of VECTASHIELD^® Vibrance™ Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The images were then observed and captured using a Nikon Ti2-E inverted fluorescent microscope along with the NIS-Element imaging software Version 5.5 (Nikon Instruments, Tokyo, Japan). The resulting images were subjected to analysis using a tally counter. The apoptotic cells were analyzed across 35 randomly chosen microscopic fields, with each group containing over 600 cells.

For immunostaining, the cultured slices were fixed with 4% paraformaldehyde in PBS at 4°C for 1 h. The fixed slices were washed with PBS and permeabilized with PBS containing 1% Triton X-100 and 5% horse serum at room temperature for 1 h. Slices were incubated with primary antibodies against GFAP (1:500) and 6His (1:200) at 4°C for 48 h. Slices were washed three times with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488 or 594 (1:500) at 4°C overnight. Slices were washed three times with PBS and then treated with VECTASHIELD Vibrance Antifade Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (#H-1800; Vector Laboratories, Newark, CA). Images were obtained using BZ-X800 (Keyence, Osaka, Japan).