Rabbit anti cx43

RBE4 cells or pBMECs were seeded in 25 cm² falcons or 8 cm² petridishes. Lysates were made with RIPA (Cx43 and Cx37) and laemmli (Cx40) buffer. Protein concentration was determined using the BioRad DC protein assay kit (BioRad, Nazareth, Belgium). The lysate was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), over a mini-protean TGX stain-free gel (BioRad, Nazareth, Belgium) and transferred to a nitrocellulose membrane (Amersham, Buckinghamshire, UK). Membranes were blocked in TBS supplemented with 5% (Cx43 and Cx40) or 2% nonfat milk (Cx37) and 1% (Cx43 and Cx40) or 0.05% (Cx37) Tween20. The following primary antibodies were used: rabbit-anti-Cx43 (sigma), rabbit-anti-Cx37 (anti-rat and anti-mouse, Alpha Diagnositcs, Reinach, Switzerland), goat-anti-Cx40 (Santa Cruz), or rabbit-anti-β-tubulin antibody (Abcam, Cambridge, UK). Membranes were subsequently incubated with an alkaline phosphatase-conjugated goat anti-rabbit (Cx43, Cx37) or donkey anti-goat (Cx40) IgG antibody (Sigma-Aldrich). Detection was done using the nitro-blue-tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate reagent (NBT/BCIP kit, Zymed, Invitrogen). Total protein staining was carried out with SYPRO Ruby protein blot dye (Invitrogen, Molecular Probes, Merelbeke, Belgium).

Cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT), permeabilised with 0.1% triton x-100 and blocked with 1% bovine serum albumin (BSA) for 1 h at RT. Cells were incubated with primary antibodies mouse anti-human α-SMA (1:100; Dako) or rabbit anti-Cx43 (1:300; Sigma-Aldrich) overnight at 4 °C, followed by AlexaFluor 488 conjugated secondary antibody (1:200; ThermoFisher) for 2 h at RT. Cells were mounted with VectaShield Propidium iodide. Images were taken with a Nikon Ti-Eclipse A1M confocal microscope using a 20×-objective. Cells positive for α-SMA were counted in 5 randomly chosen images per sample, with a minimum of 100 cells counted per sample. Cx43 was quantified with ImageJ by dividing the area of total Cx43 staining by the number of nuclei in each image.

Confluent astrocyte cultures plated in 100 mm dishes were lysed and equal amounts of total protein were loaded onto 7.5 or 10% SDS-PAGE gels for separation and electrophoretically transferred to nitrocellulose membranes (Whatman, Dassel, Germany). The membranes were probed with polyclonal antibodies to rabbit anti-Cx43, 1:10,000 (Sigma, cat# C6219); mouse anti-GFAP 1: 500 (Sigma cat# G3893), goat anti-GS 1:500 (Santa Cruz, cat# SC-6640), and goat anti-AQP4 1:500 (Santa Cruz, cat# SC-9888), followed by secondary antibody incubation with horseradish peroxidase-conjugated donkey anti-goat IgG, goat anti-rabbit IgG and goat anti-mouse (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, United States cat# SC-2020, SC-2004, and SC-2005, respectively). Protein bands were detected using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, MA, United States catalog# WBKLS0100) and images were acquired using the In Vivo FX PRO imaging system (Carestream, Carestream, NY, United States).

For Cx immunoscytochemistry (Cx37, Cx40, and Cx37), fixation was as described for γ-H2AX immunostaining, followed by a 30 min blocking step with blocking buffer B (0.2% Tx100, 0.4% gelatin). Cells were incubated overnight with sheep anti-Cx37 (1/1000, Invitrogen), goat anti-Cx40 (1/50, Santa Cruz), or rabbit-anti-Cx43 (1/500), sigma) combined with rat anti-CD31 (combination of two antibodies, each 1/100, BD and Invitrogen) at 4 °C. In a next step, secondary antibodies were administered for 1 h (donkey anti-sheep alexa 594, chicken anti-goat alexa 594, goat anti-rabbit alexa 594, and goat anti-rat alexa 488, respectively, each 1/400 in blocking buffer B). Confocal images were taken with a Leica SP8 X confocal microscope (×63 water immersion objective) and analyzed using FiJi software.