Atp6v1b1 b2

To assess protein expression, proteins isolated from each patient's wound section were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were then probed with respective primary antibodies specific for each protein: β-actin (A5316 from Sigma-Aldrich; NHE1, MCT-1 and ATP6V1B1/B2 from Santa Cruz Biotechnology, CA, USA). Dilutions were made according to the manufacturer's recommendation. After incubation with respective primary antibodies, the membranes were washed in tris-buffered saline supplemented with 0.1% Tween 20 and incubated with the appropriate secondary antibodies (Sigma-Aldrich) followed by development with the BCIP/NBT phosphatase substrate system (Pierce Biotechnology, Rockford, IL). β-actin served as loading control.

Chronic wound biopsies were fixed in paraformaldehyde and routinely processed for paraffin embedding. After blocking with 5% BSA in phosphate buffered saline supplemented with 0.1% Tween 20, sections were incubated with primary antibodies (NHE1 and ATP6V1B1/B2: Santa Cruz Biotechnology; MCT1: Millipore, Billerica, MA, USA) at room temperature for 1 h followed by several washes in PBS and incubation with fluorescently-labelled secondary antibodies, namely Alexa Fluor 594 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG (H+L) (Invitrogen). Images were acquired using an Olympus Bx63 epifluorescence microscope (100x/1.4 NA objective), and image processing (intensity adjustment only, quantitatively equal for all samples) was performed in Adobe Photoshop (Adobe Systems).