Mab5406

The following primary antibodies were used in the described experiments: mouse anti-HIP1R (612118, BD Biosciences Pharmingen), rabbit anti-HIP1R (AB9882, Milipore), mouse anti-GluN2B and rabbit anti-GFP (homemade mAb), rabbit anti-GluN2A (ab133265, Abcam), mouse anti-GluA1 (MAB2263, Millipore), rabbit anti-GluA1 (ab131507, Abcam), mouse anti-Myc (A2814, Santa Cruz), rabbit anti-Myc (ab32027, Abcam), mouse anti-MAP2 (M9942, Sigma-Aldrich), mouse anti-GAD67 (MAB5406, Millipore), rabbit anti-γ-aminobutyric acid A receptor (GABA_A)-α1 (2583376, Millipore) mouse anti-postsynaptic density (PSD95; MAB1596, Millipore), mouse anti-synaptophysin (S5768, Sigma-Aldrich), and mouse anti-β-actin (A5316, Sigma-Aldrich).
The secondary antibodies used for western blotting were goat anti-mouse IgG-HRP (31460, Pierce) and goat anti-rabbit IgG-HRP (31420, Pierce). The secondary antibodies used for immunostaining were from Life Technologies (Grand Island, NY, USA).

Juvenile, adolescent and adult rats fed a standard diet, CE-2, were perfused in the same manner as described above. The VTA-containing sections (−4.9 mm to −5.8 mm from the bregma) were washed in PBS incubated with a blocking solution (0.1% Triton-X, 0.2% BSA, 0.2% NGS) for 30 min. These sections were then incubated with a mouse monoclonal anti-GAD67 antibody (MAB5406, 1:500, Millipore, CA) overnight at 4 °C. Then, the sections were incubated with Daylight 594-labelled anti-mouse IgG (ThermoFisher, IL) for 30 min, mounted with mount medium on glass slides, and covered with a cover glass. The images were acquired using a confocal laser-scanning microscope (Fluoreview FV10i; Olympus, Osaka, Japan). All images were captures in the same light intensity. GAD-positive neurons were counted from the microscope images and the relative intensity of brightness was measured by NIH image software (Image J 1.48 v, National Institute of Health).

Immunofluorescent staining was performed as described previously (Gao et al., 2019 (link)). Tissue sections were blocked for 1 hr at room temperature in PBST (0.3% Triton X-100) with 5% normal donkey serum, followed by incubation with primary antibodies at 4°C overnight and secondary antibodies at room temperature for 2 hr in PBST (0.3% Triton X-100) with 1% normal donkey serum. Primary antibodies used for immunofluorescent staining were anti-MOR (rabbit, 1:500 for brain and spinal cord, 1:1000 for DRG, ABCAM, ab134054), anti-CGRP (goat, 1:500, ABCAM, ab36001), FITC-IB4 (1:200, Sigma, L2895), anti-c-Fos (rabbit, 1:15000, Synaptic System, 226003), anti-VGluT2 (guinea pig, 1:2000, Millipore, AB2251-I), anti-GAD67 (mouse, 1:500, Millipore, MAB5406). Secondary antibodies were donkey anti-goat IgG-Cy5, donkey anti-rabbit IgG-Cy3, donkey anti-rabbit IgG-Alexa 488, donkey anti-mouse IgG-Alexa 488 and donkey anti-guinea pig IgG-Cy5 (1:200, Jackson ImmunoResearch Laboratories).

After continuous administration of ZTC for 8 days, BrdU was firstly injected twice by 2 h interval; and after 20 days, BrdU was again injected twice by 2 h interval; and after another 24 h, the mice were sacrificed and the brain slices were prepared for immunochemistry experiment. The mouse brain was treated with 4% paraformaldehyde for more than 48 h, and then dehydrated by two steps in 20% and 30% sucrose, separately. Coronal continuous sections, thickness 20 um, each 10 pieces were collected in a small hole in a 24-well plate. Subsequently, different samples were randomly sampled at the same location and stained and the results were counted (Liang et al., 2017 (link)). BrdU staining: Sections were blocked with 1% H₂O₂ in dark at room temperature for half an hour, and melted by 2M HCI at 37°C for 1 h, and the primary antibody (1: 500; Cat. No. MAB3424, Millipore) was incubated at 4°C overnight. GAD67 staining: brain sections were blocked with 0.5% Tween in PBS for 2 h, and diluted with 0.05% Tween in PBS for primary antibody (1: 500; Cat. No. MAB5406, Millipore) and incubated overnight at 4°C. DCX brain sections were blocked with 0.5% Triton-100 in PBS for 2 h, and diluted with 0.3% Triton-100 in PBS for primary antibody (1: 500; Cat. No. 4604, Cell Signaling) and incubated overnight at 4°C.

Gelatin-embedded, 40 μm sections of PFA-fixed mouse brains were stained with the following antibodies: mouse anti-Gad67 (cat. no.: MAB5406; Millipore; 1:1000; RRID: AB_2278725); chicken anti-GFP (cat. no.: GFP-1020; Aves; 1:1000; RRID: AB_10000240); rabbit anti-NeuN (cat. no.: ABN78; Millipore; 1:1000; RRID: AB_10807945); mouse anti-parvalbumin (cat. no.: 235; Swant; 1:7000; RRID: AB_10000343); rabbit anti-calretinin (cat. no.: 7699/4; Swant; 1:5000; RRID: AB_2313763); and AlexaFluor-, Cy3- or Cy5-conjugated secondary antibodies (Invitrogen or Jackson ImmunoResearch). To ensure a more homogeneous GAD67 fluorescence for the post-hoc immunostaining of imaged brains, the brain slices were incubated twice in the primary antibody solution, each for 1 week at 4°C. The secondary antibody was also applied twice, but overnight at room temperature. Cell counting was performed manually in FIJI using the Cell Counter plug-in on confocal z-stacks. Confocal images were acquired using a Zeiss LSM700 microscope. Overview epifluorescence images were acquired using a Zeiss AxioImagerM2 equipped with a Zeiss Axiocam 503 mono camera.