Rabbit anti mef2c

Primary antibodies and dilutions used in this study: rabbit anti-Mef2c (1:500, Cell Signaling), mouse anti-mGluR2 (1:1500, Advanced Targeting Systems), mouse anti-NeuN (1:1000, Millipore), mouse anti-Calbindin (1:5000, Swant), goat anti-parvalbumin (1:2500, Swant), rat anti-GFP (1:1000, Nacalai Tesque), guinea pig anti-vGluT2 (1:2000, Millipore), rabbit anti-GFAP (1:500, Sigma Aldrich), and guinea pig anti-zebrin (1:1000, Frontier Institute). All secondary antibodies used were obtained from molecular probes and diluted 1:1000 prior to use.

Passage and donor-matched LECs and BECs were lysed with lysis buffer (25 mM HEPES, 5 mM EDTA, 1% triton-X, 150 mM NaCl, 10% glycerol, complete protease inhibitor cocktail) and then centrifuged at 13,000 rpm for 20 min at 4° to collect the supernatant. Protein concentration of lysates was determined using the Microplate BCA protein assay kit (Thermo Fisher), according to the manufacturer’s instruction. Proteins were separated by SDS-PAGE on 4–12% NuPAGE Bis–Tris protein gels (Invitrogen) and transferred to a PVDF membrane (Immobilon-P, Millipore). Membranes were blocked in 5% milk in TBS + 0.1% Tween20, and incubated with primary antibodies (rabbit anti-MEF2C 1:1000, Cell Signaling, 5030; mouse anti-BCL6, 1:500, eBioscience, GI191E; rabbit anti-b-Actin 1:5000, Abcam, ab8227; rabbit anti-H3K27me3 1:1000, Diagenode, C15410195, rabbit anti-DNMT 1:1000, Cell Signaling, 5032) in 5% milk in TBS + 0.1% Tween20, followed by washes and incubation with secondary antibodies (goat anti-rabbit and goat anti-mouse both 1:5000, Dako, labeled with HRP). Signal was developed with ECL Prime (GE Healthcare) and imaged on a ChemiDoc imaging system (Bio-Rad).

Cells were seeded into wells of a 24-well plate containing glass slides and cultured until confluence. Cells were fixed with 4% PFA for 10 min at room temperature, blocked in blocking solution (PBS, 0.05% Tween 20, 0.1% Triton-X, 5% donkey serum, 5% BSA) and stained with primary antibodies (in blocking solution) overnight at 4 °C, followed by washing in PBS and incubation with secondary antibodies for 1 h at room temperature. After washing, glass slides were mounted using Mowiol (Sigma-Aldrich). Primary antibodies were: rabbit anti-MEF2C (1:400, Cell Signaling, 5030), mouse anti-BCL6 (1:100, Santa Cruz, sc-7388) and goat anti-VE-cadherin (1:100, R&D, AF938). Secondary antibodies were: donkey anti-goat AlexaFluor594, donkey anti-mouse AlexaFluor488, donkey anti-rabbit AlexaFluor647 (1:1000, all from Life Technologies). Confocal images were taken with an LSM780 microscope (Zeiss).