Streptavidin biotin complex solution sabc kit

Two rats were used for IHC for Crym (crystallin, mu) to reveal expression pattern of Crym in A29, A30 and A23 ~ .The rats were perfused with 4% PFA and processed as described above. Sequential coronal sections containing A29, A30 and A23 ~ were immuno-stained using a method we described previously (Chen et al., 2021 (link)). Briefly, after rinses in 0.1 M of PB, the sections were incubated in 3% hydrogen peroxide solution for 10 min and in 5% bovine serum albumin (BSA) for 60 min for blocking. Next, sections were incubated at 4°C overnight with a solution containing 0.3% triton X-100 and the primary antibody [rabbit anti-Crym (PA5-65072, 1:1,000, Thermo Fisher Scientific, United States)]. Then, the sections were incubated with the secondary antibody solution (biotinylated goat anti-mouse/rabbit IgG, Boster Biological Technology, United States) followed by the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 min each. After thorough rinses, the sections were visualized by incubating in 0.1 M of PB containing 0.05% 3, 3′-diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Finally, the sections were mounted on chrome alum and gelatin-coated slides, dehydrated in gradient alcohol and xylene, and coverslipped.

The IHC for Calbindin-D28k (CB) and FG was carried out in accordance with the standard procedure to facilitate the identification of the prostriata (see Lu et al., 2020 (link)) or turn the fluorescent FG into non-fluorescent products (see Chen et al., 2020 (link)). Briefly, after rinses in 0.1 M of PB, the sections were incubated in 3% hydrogen peroxide solution for 10 min and then in 5% bovine serum albumin (BSA) for 40 min for blocking. Next, sections were incubated at 4°C overnight with a solution containing 0.3% triton X-100 and the primary antibody [mouse anti-CB (66394-1-Ig, 1:1,000, ProteinTech Group, Inc., Chicago, IL, United States) or rabbit anti-FG (AB153-I, 1:10,000, Sigma-Aldrich, St. Louis, MO, United States)]. Then, the sections were incubated with the secondary antibody solution (biotinylated goat anti-mouse/rabbit IgG, Boster Biological Technology, Pleasanton, CA, United States) followed by the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 min each. After rinses, the sections were visualized by incubating in 0.1 M of PB containing 0.05% 3,3′-diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Finally, the sections were mounted on chrome alum and gelatin-coated slides, dehydrated in gradient alcohol and xylene, and coverslipped.

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.