Ab114110

Cardiac GPs, atria and ventricles were fixed with 10% formalin and embedded in paraffin. Then, 5-μm-thick tissues were serially cut from paraffin blocks and mounted onto glass slides coated with 1% (W/V) gelatin solution. After antigen retrieval, the tissue sections were incubated with primary antibodies against Na_V1.8 α subunit (1:20, ab114110, Abcam, Cambridge, UK) at 4°C overnight. After washing three times with PBS, the sections were then incubated with the secondary antibody (HRP labeled goat anti rabbit, 1:200, ab205718, Abcam, Cambridge, UK) expression were detected using DAB (brown) staining.

Samples were lysed with RIPA buffer (Beyotime, China) and the concentration of protein was assayed using a BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, MO, USA). A total of 10 μg of protein was separated on SDS-PAGE (10 %) at 80 V for 1.5 h, and transferred onto PVDF membranes (Bio-Rad, USA) at 300 mA for 1.5 h. The following primary antibodies: Na_V1.8 (1:500, ab114110, Abcam) and β-actin (1:5000, ab8227, Abcam, Cambridge, UK) were used to incubate the blots at 37 °C for 2 h with gentle shaking. Afterwards, the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:5000; ab205718, Abcam, Cambridge, UK) was used to incubate the blots for 1 h at room temperature. The band density was analyzed using a gel imaging system and compared with an internal control.