Ab26116

IHC was performed on serial section slides in two independent multiplexed batches. The first batch followed the sequence mouse anticalcium/calmodulin-dependent protein kinase II alpha (MA1-048; Invitrogen), rabbit antisomatostatin (T-4102; Peninsula Laboratories), rabbit anti-GFP (ab290; Abcam), and mouse anti-PV (PV235; Swant). The second batch followed the sequence, mouse anti-glutamate decarboxylase (GAD67, ab26116; Abcam), mouse anti-fox-3 (NeuN, MAB 377; Millipore), and rabbit anti-GFP. After primary and secondary antibody incubation, slides were stained with DAPI. Whole slide images were captured at 20 × using an Akoya Biosciences Polaris instrument. Image exposures for each fluorochrome were constant for all slides. Detailed image analysis is described in the Supplementary Materials.

The mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and transcardially perfused with 50 mL of 0.01 M phosphate buffer saline (PBS, pH 7.4) and thereafter fixed with 4% paraformaldehyde (pH 7.4). The brains were then collected and subsequently immersed in 0.1 M PB containing 30% sucrose at 4 °C. Coronal sections via the SNr area with a thickness of 30 μm were sectioned on a cryostat (Leica CM1800; Heidelberg, Germany). Sections were incubated in a blocking PBS buffer containing 0.3% Triton X-100 and 0.05% sodium azide, 10% bovine serum albumin for 30 min at room temperature. The primary antibodies included mouse anti- Mouse anti-GAD67 (GAD1) (1:100, Cat. #ab26116, Abcam), Rabbit anti-GAD65 (GAD2) (1:50, Cat. #5843 s, CST), Rabbit anti-Parvalbumin (1:200, Cat. #80561 s, CST), Rabbit anti-vGlut1 (1:500, Cat. #a12879, Abclonal), Rabbit anti-GFAP (1:200, Cat. #12389 s, CST), and Rabbit anti-NeuN (1:50, Cat. #ab190565, Abcam). The secondary antibodies Alexa 488-Goat anti-Mouse (1:500, Cat. #A23210, Abbkine) and Alexa 488-Goat anti-Rabbit (1:500, Cat. #A23220, Abbkine) were used, together adding DAPI (1:500, Cat. #BMD0063, Abbkine) at room temperature for 12 min. All images were taken under FV-1000 confocal fluorescence microscope. There were 3 mice in each group and at least 4 slices were taken from each brain tissue.

The primary visual cortex of adult mice was dissected under deep anesthesia with sodium pentobarbital (83 mg/kg, i.p.). The bilateral cortices of each mouse were mixed as one sample. Proteins were extracted with RIPA lysis buffer containing 0.01 M PBS (pH 7.4), 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, and complete EDTA-free protein inhibitor cocktail (Roche Applied Science). After blocking in 5 % nonfat dry milk (wt/vol, in 20 mM TBST), the membrane was incubated with rabbit anti-NR2A (1:1000, Abcam, ab77980), rabbit anti-NR2B (1:1000, Cell Signaling Technology, 4212S), rabbit anti-GAD65 (1:2000, Proteintech, 20746-1-AP), mouse anti-GAD67 (1:1000; Abcam, ab26116), and mouse anti-β-actin (1:15,000; Abcam, ab6276) antibodies, followed by the respective HRP-conjugated secondary antibodies (1:2500-1:10,000; Promega). The membrane was then developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Western blot analysis was repeated multiple times, and the optical density of each band was determined using ImageJ software and normalized to that of β-actin.

According to the specific location of the PVN in the rat brain atlas, transverse sections with a thickness of 18 μm were obtained and immunofluorescence staining was performed [34 (link)]. The primary antibodies were SIRT1 (#7475S, CST, MA, USA, 1:400 dilution), NOX4 (ab-133303, Abcam, Cambridge, UK, 1:1000 dilution), TH (sc-25269, Santa Cruz, TX, USA, 1:50 dilution), and GAD67 (ab-26116, Abcam, Cambridge, UK, 1:200 dilution). Dihydroethidium (DHE, Molecular Probes, Eugene, OR, USA) was used to examine ROS generation.

Mice were intracardially perfused with 0.9% saline followed by 4% paraformaldehyde after deep anesthesia by sevoflurane. Mouse brains were harvested and fixed in 4% paraformaldehyde for 6–8 h, and then the brains were subsequently dehydrated in 30% sucrose for 3 days. Using a freezing microtome (CM1950, Leica), 20‐μm‐thick coronal sections of the mouse brains were prepared. The brain sections were then blocked with 10% goat serum after the membranes were ruptured with 0.8% PBST. For immunofluorescence staining, the brain sections were incubated at 4°C with primary antibodies for 3 days and nights. The primary antibodies used were as follows: anti‐BDNF (1:100, ab108319, Rabbit, Abcam) mixed with anti‐GAD67 (1:300, ab26116, Mouse, Abcam); anti‐BDNF mixed with anti‐CamKIIα (1:300, 50049S, Mouse, Cell Signaling Technology); and anti‐c‐Fos (1:400, 2250, Rabbit, Cell Signaling Technology) mixed with anti‐GAD67 and anti‐c‐Fos mixed with anti‐CamKIIα. Afterward, the brain sections were probed with fluorescent secondary antibodies (1:400, ab150080, goat anti‐rabbit; ab150113, goat anti‐mouse; ab150115, goat anti‐mouse, Abcam) for 1 h at 37°C. Finally, DAPI (ab104139, Abcam) was used to stain the nucleus, and the staining outcomes were viewed using a confocal microscope (FV1000, Olympus).

After mice were perfused with 0.9% saline following by 4% paraformaldehyde under sevoflurane anesthesia, the mice brains were harvested, fixed in 4% paraformaldehyde for 6–8 h, and dehydrated in 30% sucrose for 3 days. Using a frozen microtome (CM1950, Leica), the mice brains were cut into 30‐μm‐thick coronal sections. After antigen retrieval, the brain sections were ruptured of membranes with 0.8% PBST and then blocked with 5% goat serum. The brain sections were incubated with primary antibodies for 3 days and nights at 4°C. We used the following primary antibodies for immunofluorescence: anti‐SIRT1 (1:100, 9475S, Cell Signaling Technology) mixed with anti‐GAD67 (1:300, ab26116, Abcam) and anti‐SIRT1 mixed with anti‐CamKIIα (1:300, 50049S, Cell Signaling Technology). Thereafter, the brain sections were probed with fluorescent secondary antibodies (1:400, ab150080, ab150113, ab150115, Abcam) for 1 h at 37°C. The nucleus was stained with DAPI (ab104139, Abcam). A confocal microscope was used to view the staining outcomes (FV1000, Olympus).