Acc 021

One hundred micrograms proteins were loaded to 7.5% polyacrylamide gel and transferred to 0.45 μm PVDF membranes (Millipore). Membranes were blocked with 5% (w:v) milk for 1 h at room temperature then incubated with primary antibodies at 4 °C overnight. The membranes were washed with TBST three times and incubated with secondary antibody for 1 h at room temperature. Finally, the membranes were developed with Clarity Western ECL Substrate (Bio-Rad) and pictures were taken by ChemiDoc Touch (Bio-Rad). Antibodies used were anti-TRPC7 1:500 (HPA031126, Sigma), anti-β-tubulin 1:1000 (15,115, Cell Signaling, Danvers, Massachusetts, USA), anti-HCN4 1:200 (APC-052, Alomone), anti-Cav1.3 1:100 (ACC-005, Alomone), anti-Cav3.1 1:200 (ACC-021, Alomone), anti-Cav3.2 1:200 (ACC-025, Alomone), anti-RyR2 1:1000 (MA3-916, Invitrogen), anti-SERCA 1:200 (sc-30110, Santa Cruz), anti-IP3R 1:500 (ACC-019, Alomone), anti-p(S2814)RyR2 1:5000 (A010-31, Badrilla), anti-phospholamban (PLN) 1:1000 (A010-14, Badrilla), anti-p(T17) PLN 1:5000 (A010-13, Badrilla), HRP-conjugated goat anti-rabbit secondary antibody 1:5000 (Dako, Zug, Switzerland), and HRP-conjugated goat anti-mouse secondary antibody 1:5000 (Dako).

Immunohistochemistry was performed using the peroxidase reaction technique with identification by polymerized secondary antibody (Novocastra Post Primary and Novolink Polymer; Leica Biosystems, UK). The antigen retrieval was performed by pressurized humid heat at 137°C (Autoclave ALT 5LD Plus; ALT, Brazil) with Target Retrieval Solution Citrate - low pH (Agilent Technologies, USA). Blockage of endogenous peroxidase and endogenous proteins was done with Novocastra reagents (Leica Biosystems). Antibodies anti-Ca_V3.1, -Ca_V3.2, and -Ca_V3.3 (ACC-021, ACC-025, and ACC-009; Polyclonal, Alomone Labs, Israel) were used in dilutions of 1:250, 1:100 and 1:200, respectively. Incubation with primary antibody was set at 16 h and with 3'3-diaminobenzidine chromogen Novocastra DAB Chromogen (Leica Biosystems) at 1 min. Sections were counterstained with hematoxylin dye. For positive controls of Ca_V 3.1 and Ca_V3.2 labelling, we used samples of human skeletal muscle, and for Ca_V3.3, samples of human brain were used. For negative controls, the primary antibody incubation step was replaced by incubation with immunoglobulins of the same species as the primary antibody.