Cldn2

RT was performed by using iScript Reverse Transcription Supermix kit (Bio-Rad). PCR reactions were run on a CFX96 Touch Real Time PCR (Bio-Rad) with SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). Primers for qPCR (Cldn2, Dlx1, Dlx5, Etv5, Gabrb1, Gabrg2, Gabra3, Gad2, Gapdh, Itgb3, Lhx1, Lhx6, Mef2c, Nkx2.1, Nkx2.2, Nrp1, VegfA, Wnt10a, Wnt10b) were obtained from Thermo Fisher Scientific. The housekeeping gene Gapdh was used as a reference. The relative gene expression among different samples and subsequent fold increase in Vgat^fl/fl versus Vgat^ECKO endothelial cells or GABAergic neurons was determined according to published methodology^{44 (link)}.

The qPCR-based quantification of the gene expression levels of the cDNA samples was performed using a TaqMan®-based singleplex assay with Actb (Mm00607939_s1) as the endogenous control gene, Mki67 (Mm01278617) and Slc5a1 (Mm00451210_m1) as the target, and the following TaqMan®-based multiplex assays: 4-plex 1 [Actb (Mm00607939_s1_qsy_ABY) as the endogenous control gene, Cldn2 (Mm00516703_s1_VIC; data not shown), Cldn7 (Mm00516817_m1_qsy_JUN; data not shown), and Tnfα (Mm00443258_m1_FAM)] and 4-plex 2 [Cldn4 (Mm_00515514_s1_qsy_ABY), Cldn8 (Mm00516972_s1_qsy_JUN; data not shown), Ocln (Mm00500912_m1_FAM), and Tjp1 (Mm01320638_m1_VIC)] (all from Thermo Fisher Scientific, Waltham, MA, USA). SYBR® Green-based QuantiTect Primer Assays (Qiagen®, Hilden, Germany) were used for Actb (Mm_Actb_1_SG) as the endogenous control gene, and Chga1 (Mm_Chga_1_SG) and Muc2 (Mm_Muc2_2_SG) as target genes. Each sample was either measured in duplicate or triplicate using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA). Relative quantification (RQ) was performed using the 2^−ΔΔCT method [16 (link)].