Ab153 1

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.

Retrograde tracer injections were performed on adult wild-type littermates of Rbp4–Cre mice (n = 4). Fluorogold (Sigma-Aldrich, AB153-I) was injected iontophoretically (0.5 μA; 2-second ON/OFF period, 10-minute duration) in the right side of the thalamus using borosilicate glass capillaries. Stereotaxic coordinates were the following: VM: AP −1.3, ML 0.8, DV −4.2; MD: AP −1.3, ML 0.5, DV −2.9.

Sections from the brains injected with FG were examined under an epifluorescent microscope (Leica DM6B) or stained with immunohistochemistry (IHC) according to standard procedures. For the IHC, the sections were rinsed in 0.1M PB three times, for 10 min each time, and then incubated in room temperature with 3% hydrogen peroxide for 10 min. After blocking in 5% BSA for 40 min the sections were incubated at 4°C overnight with solution containing 0.3% triton X-100 and primary antibody (rabbit anti-FG, AB153-I, 1:10000, Sigma-Aldrich). After that, the sections were incubated with the secondary antibody solution (biotinylated goat anti-mouse/rabbit IgG, Boster Biological Technology) and then the Streptavidin-Biotin Complex solution (SABC kit, Boster Biological Technology) for 60 min each. The sections were visualized by incubating the sections in PB containing 0.05% 3, 3-diaminobenzidine (DAB) and 0.01% hydrogen peroxide. Finally, the sections were rinsed and mounted on chrome alum and gelatin-coated slides, dehydrated in a graded series of ethanols, and coverslipped.