Acc 120

Immunofluorescence detection of proteins was performed as described previously [27 (link)]. Briefly, kidneys were perfused and fixed in 4% PFA for 2 h at room temperature, embedded in paraffin, and sectioned 8 μm thick. For immunofluorescence analysis of antibody stains, slides were washed in 1XPBS. For antigen retrieval, heat-induced epitope retrieval with Sodium Citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) was applied. To reduce non-specific binding, sections were blocked with normal serum (1:20). Primary and secondary antibodies were applied sequentially overnight at 4° C and 2 h at room temperature. Primary antibodies and dilutions were as follows: anti-TRPC6 antibody (1:100, ACC-120, Alomone Labs, Jerusalem, Israel), anti-P2Y2 antibody (1:100, APR-010, Alomone Labs, Jerusalem, Israel), anti-GFP antibody (GFP 1:200 Thermo Fisher Scientific, Waltham, MA). Alexa Fluor 488 and 594-conjugated secondary antibodies were purchased from Invitrogen (Waltham, MA). Slides were mounted by using DAPI-containing mounting media (VectaShield, Vector Laboratories Inc., Burlingame, CA). Sections were examined with Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) confocal/multiphoton laser scanning microscope systems as described previously [27 (link), 28 (link)].