Na2po4

Human differentiated podocytes were cultured on glass coverslips in 6‐well plates, were fixed by acetone for 5 min. at room temperature, and permeabilized by 0.6% Triton‐X‐100 (Sigma‐Aldrich) in PBS (115 mM NaCl, 20 mM Na₂PO₄, pH 7.4; all from Sigma‐Aldrich) for 10 min. Following 30 min. incubation in blocking solution [0.6% Triton‐X‐100 and 1% bovine serum albumin (BSA) containing PBS; Sigma‐Aldrich] at room temperature, cells were probed with the previously mentioned primary antibodies raised against TRPC6 (1:50), podocin (1:100) and synaptopodin (1:100) overnight at 4°C. Following appropriate washing in PBS, coverslips were incubated with Alexa‐488^®‐conjugated goat anti‐mouse and goat anti‐rabbit secondary antibodies (1:200, Invitrogen) for 1 hr at room temperature. Nuclei were counterstained with propidium‐iodide (Vector Laboratories, Peterborough, UK). Negative control cells were stained omitting the primary antibodies. Visualization of the proteins was performed using Zeiss LSM 510 Meta Confocal Microscope (Zeiss, Oberkochen, Germany). The exposure time and all other settings (gain, gamma and intensity of the excitation) were exactly the same in all cases, including the negative controls.

Tissue was harvested using our well-established two-stage perfusion protocol allowing for the harvest of both live tissue and fixed tissue (Amatrudo et al., 2012 (link)) for parallel experiments not in the present study. After sedation with ketamine hydrochloride (10 mg/kg) the monkeys were deeply anesthetized with sodium pentobarbital (to effect, 15 mg/kg, i.v), and then perfused through the ascending aorta with ice-cold Krebs–Henseleit buffer containing (in mM): 6.4 Na₂HPO₄, 1.4 Na₂PO₄, 137 NaCl, 2.7 KCl, 5 glucose, 0.3 CaCl₂, and 1 MgCl₂, pH 7.4 (Sigma-Aldrich). Fresh tissues were collected from the left hemisphere for parallel biochemical and electrophysiological studies not in the present study. Once the fresh tissue harvest was complete, the perfusate was switched to 4% paraformaldehyde in 0.1M phosphate buffer (PB, ph 7.4, at 37°C) to fix the remaining whole brain. The fixed brain sample was blocked, in situ, in the coronal plane, removed from the skull, cryoprotected in a series of glycerol solutions, and flash-frozen in −70°C isopentane (Rosene et al., 1986 (link)). The brain was cut on a freezing microtome in the coronal plane at 30 or 60 μm and stored in cryoprotectant (15% glycerol, in 0.1M PB, pH 7.4) at −80°C (Estrada et al., 2017 (link)).

Osteogenic induction medium was composed of 10% FBS-αMEM supplemented with 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, United States), 10 mM β-glycerophosphate (Sigma-Aldrich) and 50 μM L-ascorbic acid 2-phosphate (Sigma-Aldrich) (Jaiswal et al., 1997 (link)). For each sample, 5 × 10⁴ cells per cm² were seeded into a 6-well plate. After reaching confluence in 2 days, the cells were incubated with osteogenic induction medium. The medium was changed every 3 days, and analyses were performed after 14 days of induction. To assess calcium precipitation, the induction medium was removed, and the cells were washed once with PBS. Next, the cells were fixed with 10% formaldehyde (Sigma-Aldrich) for 10 min with gentle shaking, and after another wash with PBS, the cells were stained with 500 μL of 2% ARS (pH 4.1–4.3) (Sigma-Aldrich) for 15 min. After extensive PBS irrigation, the precipitate was dissolved in 500 μL of 10% cetylpyridinium chloride (Sigma-Aldrich) in 8 mM Na₂PO₄ (Sigma-Aldrich) and 1.5 mM KH₂PO₄ (Sigma-Aldrich). The absorbance at 550 nm was recorded, and quantification of calcium was determined using an optimal ARS standard curve. The optimal dilution was performed to ensure that the sample absorbance fell within the standard curve.