Staining Protocol for BDA-Labeled Neurons

The sections from the brains injected with BDA were stained according to a previously published method (Chen et al., 2021 (link); Xiang et al., 2023 (link)). Briefly, the sections were first washed in 0.05 M PBS (at least three times, 5 min each), and incubated in the Triton solution (0.3% Triton X-100:0.05 M PBS = 3:1,000) at room temperature for 1 h. The sections were then incubated in a Streptavidin-Biotin complex solution (SABC kit, Boster Biological Technology) for 3 h at room temperature. After rinsing in 0.05 M PBS three times, the sections were visualized with 0.05 M PBS containing 0.05% 3, 3-diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Finally, the sections were mounted on chrome alum and gelatin coated glass slides, dehydrated in gradient ethanol and xylene, and finally coverslipped.