Biotinylated goat anti mouse rabbit igg

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 sagittal sections containing the major downstream target regions of the rat prostriata were selected for c-fos IHC. These regions include the PrSd-PoS, LD, LP-Pul, PTN, and VLG and are directly innervated by the prostriata (Chen et al., 2021 (link)). In addition, we also evaluated c-fos expression in zona incerta (ZI) and substantia nigra (SN), which are not the targets of the prostriata. Selected sections were rinsed three times with 0.1 M PB and immersed in 0.3% hydrogen peroxide for 10 mins. Then the sections were rinsed three times again and blocked in 5% BSA for 60 mins. At the end, the sections were incubated with primary antibody against c-fos (200 μg/ml, 1:200, Boster Biological Technology, Wuhan, China) diluted with the 0.1 M PB overnight at 4°C. On the next day, the sections were incubated in the secondary antibodies (biotinylated goat anti-mouse/rabbit IgG, Boster) for 60 mins after thorough rinse with 0.1 M PB. The sections were then rinsed again and immersed in the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 mins. After rinse with 0.1 M PB, the sections were incubated in 3, 3-diaminobenzidine (DAB) solution for 3 mins in a dark environment. Finally, the sections were mounted on the slides, dehydrated 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.