Goat anti cx40

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).

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.