Positive fluorescence microscope

Immunofluorescence assays were conducted as previously described [12 (link)]. Briefly, BCa cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-100 in PBS, blocked with 5% BSA (bovine serum albumin) and incubated with the indicated primary antibodies dissolved in PBS 1:200 at 4 °C overnight. After incubation with the fluorescent secondary antibody at room temperature for 1 h, staining with DAPI for 5 min in the dark and sealing with glycerin, the stained images were captured under a positive fluorescence microscope (Olympus, Tokyo, Japan). The experiments were performed at least in triplicate.

The proliferation ability of BCa cells was detected by EdU assay using the EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, China). Cells were washed with PBS three times and incubated with complete medium with 10 μM EdU in a cell incubator for at least 2 h. Then, the cells were washed with PBS to remove the EdU probe and culture medium, fixed with 4% paraformaldehyde for 10 min at room temperature, and stained with DAPI for 5 min in the dark. The cells were observed under a positive fluorescence microscope (Olympus, Tokyo, Japan) at 100x magnification in five random fields. The staining signals were captured, analyzed, and shown as fold changes compared with the control. The experiment was repeated three times.

As described in the previous study [16 (link)], brain tissues were obtained after transcardial perfusion, and TUNEL/NeuN staining and Nissl staining were conducted. The Golgi-Cox staining was performed using the FD Rapid GolgiStain Kit following the manufacturer’s instructions (FD NeuroTechnologies, Columbia, MD, USA). 6 random high-power fields were chosen and images were collected using a positive fluorescence microscope (Olympus, Osaka, Japan).