Anti cnx43

A xylene substitute (HEMO‐De; Fisher Scientific) was used to remove paraffin from tissue sections and then rehydrated with ethanol gradient washes. A 3% hydrogen peroxide aqueous solution was used to sequentially quench the tissue sections, blocked for 1 hour at room temperature with phosphate‐buffered saline (PBS) 6% non‐fat dry milk (Bio‐Rad) and then incubated with specific antibodies anti‐Cnx43 (diluted 1:100; Santa Cruz; Cat. No. sc‐271837) and anti‐KCNQ1 (diluted 1:200; LifeSpan Biosciences; Cat. No. LS‐C405102), according to the recommended dilutions. After PBS washes, sections were incubated with secondary biotin‐conjugated goat anti‐rabbit IgG and avidin–biotin peroxidase complex (diluted 1:200; DBA; Cat. No. BA‐1000). An expert pathologist, blinded to the experimental protocol, analysed the specimens (intra‐observer variability 6%). The antigen expression was automatically calculated by using Image program LEICA IM500 and LEICA QWIN statistic program. Five distinct preparations for each group of animals were carried out, by analysing in each of them 20 microscope fields, for a total area of 2.4755 e⁺⁰⁰⁴ at 400× magnifications.

Thirty microgram of protein sample was separated on a 12% separation gel, electrotransferred onto a polyvinylidenedifluoride (PVDF) membrane (Merck Millipore; Cat. No. IPFL10100) and then blocked for 1 hour at room temperature with 5% non‐fat dry milk (Euroclone; Cat. No. EMR180500). The following specific primary antibodies were used for the overnight incubation of the blots using the recommended dilutions: anti‐HIF‐1α (diluted 1:200; Santa Cruz; Cat. No. sc‐8711), anti‐Cnx43 (diluted 1:500; Santa Cruz; Cat. No. sc‐271837), anti‐KCNQ1 (diluted 1:200; LifeSpan Biosciences; Cat. No. LS‐C405102) and anti‐actin (diluted 1:500; Santa Cruz; Cat. No.sc‐8432). Blots were then incubated for 1 hour at room temperature with horseradish peroxidase‐conjugated secondary anti‐rabbit (diluted 1:2000; Santa Cruz; Cat. No. sc‐2004), anti‐goat (diluted 1:2000; Santa Cruz; Cat. No. sc‐2020) or antimouse (diluted 1:2000; Santa Cruz; Cat. No. sc‐2005) antibodies. The signal was detected using the VisionWorks^™ LS Analysis Software (Analytik Jena AG) and then expressed as densitometric units (DU).