Anti vglut2

Animals were anesthetized with sodium pentobarbital, and perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (PBS). Brains were removed, post-fixed for 5–6 h in 4% PFA, and then transferred to 30% sucrose and kept at 4 °C for 2 days. Sagittal sections were cut at 40 µm on a microtome (Cryostar NX50, Thermo, Waltham, USA). After washing three times with 0.01M PBS, rinsing with frozen methanol (10 min at -20 °C), and blocking with 10% bovine serum albumin (BSA) for 1 h at room temperature, the sections were incubated with primary antibodies as follows: anti-VGluT2 (guinea-pig polyclonal, 1:600, Millipore, Billerica, USA, Cat# AB2251-I, RRID: AB_2665454) and anti-NeuN (rabbit monoclonal, 1:500; Millipore Cat# MABN140, RRID: AB_2571567) at 4 °C for 12–24 h. After rinsing, secondary fluorophore-conjugated antibodies (Alexa Fluor 488, donkey anti-rabbit, 1:1000, Thermo Fisher Scientific Cat# A-21206, RRID: AB_2535792; Alexa Fluor Cy3, donkey anti-guinea pig, 1:1000, Jackson, West Grove, USA, Cat# 706-165-148, RRID: AB_2340460) were applied for 2 h at room temperature. The antibodies were diluted in PBS containing 5% BSA. Images were captured using a 60× objective on an Olympus FV-1000 or FV-1200 inverted confocal microscope. 10–12 consecutive images at 0.5 mm intervals were captured in Z-stacks.

The mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and sequentially perfused with saline and 4% (w/v) paraformaldehyde (PFA). The brains were subsequently removed and post-fixed in 4% PFA at 4 °C overnight. After cryoprotection of the brains with 30 % (w/v) sucrose, coronal sections (40 μm) were cut on a cryostat (Leica CM1860, Germany) and used for immunofluorescence. The sections were firstly treated with citrate antigen retrieval solution, and then incubated in 0.3 % (v/v) Triton X-100 for 0.5 h, blocked with 5% donkey serum for 1 h at room temperature, and incubated with primary antibodies, including anti-MeCP2 (1:500, rabbit, Cell Signaling) and anti-vGluT2 (1:50, mouse, Millipore) at 4 °C for 24 h, followed by the corresponding fluorophore-conjugated secondary antibodies, including Alexa Fluor 488 donkey anti-rabbit IgG (1:500, Invitrogen), Alexa Fluor 594 donkey anti-mouse IgG (1:500, Invitrogen), and Alexa Fluor 594 donkey anti-rabbit IgG (1:500, Invitrogen) for 2 h at room temperature. Immunofluorescence staining for MeCP2, vGluT2 and their overlap from randomly selected sections (n = 3–4 sections from each mice). Fluorescence signals were visualized using a Zeiss LSM710 microscope, and further analyzed using TissueQuest software (TissueGnostics, US).