Trek 1

Human TM sections embedded in paraffin were deparaffinized, hydrated for 20 min with 1X phosphate-buffered saline (PBS) (cat# 21-040-CV, Mediatech Inc., Manassas, VA), and blocked in 1X PBS + 0.2% bovine serum albumin (BSA) (Fraction V, cat# 2910, EMD Chemicals, Gibbstown, NJ) for 30 m. The primary antibody was added for cochlin and TREK-1 in 1:200 dilution (cochlin: hCochlin #3 (cat# 5007/5008) Aves Labs Inc., TREK-1: cat# ab83932, Abcam, Cambridge, MA). After incubating overnight at 4 °C, primary antibody was washed out with 1X PBS + 0.2% BSA 3 times for 10 m per wash. The corresponding secondary antibody was added in 1:1000 dilution (Alexa594, cat# A11042, Invitrogen; Alexa488: cat# ab150073, Abcam) and incubated for 1 h at room temperature. The sections were then mounted on glass microscope slides (cat# 48300-0205, VWR International, West Chester, PA) and stained with DAPI Vectashield (cat# H-1200, Vector Laboratories). Thus prepared slides were imaged using Leica DM 6000 B confocal microscope (Leica, Inc.).

Protein detection was performed in RIPA buffer (Sigma) lysed VIC. Total protein levels were quantified with a Pierce BCA protein assay (Thermo Scientific, Hemel Hempstead, UK). Protein lysates (7.5 μg) were electrophoretically separated under denaturing conditions on 10% Bis-Tris gels (Invitrogen, Renfrew, UK), and transferred on to nitrocellulose membranes (Hybond C, Amersham, UK). Specific MSC were detected using following antibodies: Kir6.1 (Bioss, London, UK), TREK-1, TRPM4, TRPV4, TRPC6 (Abcam, Cambridge, UK), followed by washing and incubation with secondary antibodies. Bands were visualized using enhanced chemiluminescence substrate and positivity was captured on Hyperfilm (GE Healthcare, Amersham, UK). Films were scanned and bands were quantified using the QuantityOne program (Biorad, Hercules, USA). Levels of protein expression were normalised to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (R&D Systems, Abingdon, UK).