Anti cytokeratin antibody

Mouse kidney TECs were isolated and cultured as described previously^{65 (link)}. In brief kidneys were perfused with saline then removed. Kidney cortices were dissected into approximately 1 mm³ pieces and digested in HBSS containing 3 mg/mL of collagenase at 37 °C for 25 minutes, followed by washing in DMEM/F12 medium (Invitrogen). The kidney digest was washed through a series of sieves (mesh diameters of 250, 150, 75 and 40 µm) then spun down at 300 g for 5 minutes. The cell pellet was re-suspended in defined K1 medium: DMEM/F12 supplemented with 25 ng/mL epidermal growth factor, 1 ng/mL PGE1, 5 × 10-11 M triiodothyronine, 5 × 10-8 M hydrocortisone (Sigma-Aldrich), ITSS media supplement, 1% penicillin/streptomycin, 25 mM HEPES, and 5% FCS (Invitrogen). The cell suspension was then seeded on cell culture Petri dishes and incubated at 37 °C for 2–3 hours to facilitate adherence of contaminating glomeruli. The non-adherent tubules were collected and cultured on collagen-coated Petri dishes (BD Biosciences) in K1 medium. Expression of the epithelial cell marker cytokeratin was verified by immunofluorescent staining with an anti-cytokeratin antibody (Sigma-Aldrich). Cells were >95% cytokeratin positive. Experiments were commenced after cells had reached 80% confluence.

Embryonic kidneys were dissected in ice-cold PBS, fixed in 4% PFA 1 h at 4 °C, then washed and stained with an anti-cytokeratin antibody (Sigma) at a 1:250 dilution in 1× PBS supplemented with 5% goat serum (Cat#16210-064, Life Technologies) for 48 h at 4 °C, then washed and stained using an Alexa Fluor 555 goat anti-mouse IgG1 fluorescent secondary antibody (Cat#A21127, Molecular Probes) at a 1:125 dilution in 1× PBS/5% goat serum overnight at 4 °C, then washed again with PBS and processed for OPT as follows. Stained embryonic kidneys were embedded in 1% low melting point agarose, such that the tissue was completely surrounded by agarose. Blocks were trimmed to remove excess agarose, dehydrated in 50% methanol for 6 h, transferred to 100% methanol for 24 h, cleared overnight in a 2:1 mixture of benzyl alcohol/benzyl benzoate and then mounted on a metal OPT magnet, and imaged in a Bioptonics 3001 OPT scanner (Bioptonics, Edinburgh, UK), at maximum resolution of 3.1 μm per pixel zoom. Images were acquired at 0.90° intervals. Post-alignment and 3D reconstruction (filtered back projection method) were performed using NRecon software (Skyscan).

Mouse kidney TECs were isolated and cultured from WT C56BL/6, Gpr43−/− and Gpr109A−/− mice as described previously (Li et al., 2020 (link)). In brief, kidneys were perfused with saline then removed. Kidney cortices were dissected into 1 mm³ pieces and digested in HBSS containing 3 mg/mL of collagenase at 37°C for 25 min, followed by washing in DMEM/F12 medium (Invitrogen). The kidney digest was washed through a series of sieves (mesh diameters: 250, 150, 75 and 40 µm) then spun down at 300 g for 5 min. The cell pellet was re-suspended in defined K1 medium: DMEM/F12 supplemented with 25 ng/mL epidermal growth factor, 1 ng/mL PGE1, 5 x 10-11M triiodothyronine, 5 x 10-8M hydrocortisone (Sigma-Aldrich), ITSS media supplement, 1% penicillin/streptomycin, 25 mM HEPES, and 5% FCS (Invitrogen). Cell suspension was then seeded on cell culture Petri dishes and incubated at 37°C for 2–3 h to facilitate adherence of contaminating glomeruli. The non-adherent tubules were collected and cultured on collagen-coated Petri dishes (BD Biosciences) in K1 medium. Expression of the epithelial cell marker cytokeratin was verified by immunofluorescent staining with an anti-cytokeratin antibody (Sigma-Aldrich). Cells were >95% cytokeratin positive.