Anti cx43 c6219

Frozen left ventricular tissue was homogenised in lysis buffer of 20% SDS, 10 mmol/L EDTA, 100 mmol/L Tris and pH 6.8. This was diluted in Laemmli buffer. Equal amounts of protein per lane were separated in 10% SDS-polyacrylamide gels at 120 V (Mini-Protean Tetra Cell, Bio-Rad, Hercules, CA, USA). The proteins were transferred to a nitrocellulose membrane (0.2 m pore size, Advantec, Tokyo, Japan) and blocked for 4 h with 5% low-fat milk prior to overnight incubation at 4 °C with the following primary antibodies; anti-Cx43, C6219 Sigma-Aldrich, Saint-Louis, MO, USA; anti-PKCε, sc-214; anti-PKCδ, sc-213; anti-GAPDH, sc-25778—all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. The membranes were washed with Tris-buffered 0.1% Tween (TBS-T) and incubated for 1 h with a horseradish peroxidase-linked secondary antibody (anti-rabbit, diluted 1:2000, Cell Signalling Technology, Danvers, MA, USA, #7074), then washed in TBS-T and visualised by enhanced chemiluminescence. Finally, the Protein bands were quantified by version 5.0 Carestream molecular imaging software densitometric analysis (Carestream Health, New Haven, CT, USA) and normalisation to the GAPDH band. The representative immunoblots (proteins and GAPDH) in figures were spliced to allow better comparison. The spliced blots were from the same gel.

Paraformaldehyde-fixed, paraffin-embedded bladders from control and uCx43KO mice were treated with citrate buffer for antigen retrieval, and immunohistochemistry was performed by the avidin–biotin complex (ABC) method. Anti-Cx43 (C6219, Sigma-Aldrich, St. Louis, MO, USA, 1:500) was used as the primary antibody against Cx43, and the biotinylated secondary antibodies (1:300) and the ABC kit were used following the manufacturer’s instructions (ABC-Elite, Vector Laboratories, Burlingame, CA, USA).

Hearts were explanted from isoflurane anesthetized rats and homogenized as previously described [20 (link)]. Ten percent Bis-Tris gels and MOPS running buffer were used and the proteins were blotted onto nitrocellulose membranes (all from Life technologies, Nærum, Denmark). Anti-Cx43 (C6219) 1:50.000, anti-β-tubulin (T0198) 1:2000 (both from Sigma-Aldrich, Brøndby, Denmark), anti-mouse (IRdye680RD) 1:10.000, and anti-rabbit (IRdye800CW) 1:10.000 (LI-COR Biosciences, Cambridge, UK) were used and membranes analyzed using Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Cambridge, UK).

For flow cytometric analysis, anti-CD45.2-V500(562129, BD bioscience, 1:100), anti-CD45.2-APC(109814, BioLegend, 1:100), anti-CD4-PerCP/Cy5.5(100433, BioLegend, 1:100), anti-CD8a-PerCP/Cy5.5(100733, BioLegend, 1:100), anti-CD11b-PerCP/Cy5.5(45-0112-80, eBioscience, 1:100), anti-Ly6G-PerCP/Cy5.5(127615, BioLegend, 1:100), anti-B220-PerCP/Cy5.5(103235, BioLegend, 1:100), anti-CD11b-BV421(101251, BioLegend, 1:100), anti-CD64-APC(139306, BioLegend, 1:100), anti-F4/80-PE(123110, BioLegend, 1:100), anti-Ly6c-PE-Cy7(128018, BioLegend, 1:100) were used. For western blotting, anti-Cx40(AB1726, Merck Millipore, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-Cx45(AB1745, Merck Millipore, 1:2000), anti- α -tubulin(T6199, Sigma, 1:1000), anti-rabbit IgG-HRP(7074, Cell Signaling Technology, 1:5000), anti-mouse IgG-HRP(7076, Cell Signaling Technology, 1:5000) were used. For immunohistochemistry, anti-F4/80(MCA497G, Serotec, 1:500), anti-Ly6G(127601, BioLegend, 1:500), anti-B220 (103201, BioLegend 1:500), anti-CD3(GTX42110, GeneTex, 1:500), anti-Cx43(C6219, Sigma, 1:2000), anti-N-cadherin(33-3900, ThermoFisher, 1:500) were used. The second antibodies used were Alexa-Fluor-488-conjugated anti-mouse-IgG(A-11001, ThermoFisher, 1:2000) and Alexa-Fluor-635-conjugated anti-rabbit-IgG(A-31576, ThermoFisher, 1:2000).