Ab212197

For GFP immunostaining, 4% paraformaldehyde-fixed renal cryosections (8 μm) from Piezo2^GFP mice and wild-type mice were pretreated with proteinase type XIV (0.02–0.05%, Sigma, Tokyo, Japan) for 10 min, incubated with 0.5% blocking reagent (Roche Diagnostics), then incubated with a chicken anti-GFP antibody (A10262, 1:100; Thermo Fisher Scientific) overnight at 4 °C, and finally incubated with an Alexa Fluor 488-conjugated anti-chicken IgY (H + L) secondary antibody (A11039, 1:200; Thermo Fisher Scientific)^{23 (link),28 (link)}. For double immunostaining, GFP-stained sections were incubated with a rabbit anti-Pdgfrb (3169S, 1:100; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Nphs2 (29040, 1:100; Immuno-Biological Laboratories, Gunma, Japan), rat anti-Pecam1 (553370, 1:200; BD Biosciences, Franklin Lakes, New Jersey, USA), or rabbit anti-Ren1 (ab212197, 1:1000; Abcam, Cambridge, UK), followed by an Alexa Fluor Plus 555-conjugated anti-rabbit IgG (H + L) secondary antibody (A32794, 1:200; Thermo Fisher Scientific) or Cy3-conjugated Affini Pure F(ab′)₂ fragment anti-rat IgG (H + L) secondary antibody (712-166-153, 1:200; Jackson ImmunoResearch, West Grove, PA, USA). Fluorescence signals were observed using confocal laser scanning microscope (LSM 980; Zeiss).

Cells were gently washed twice with PBS, fixed with 4% PFA for 15 min, washed with PBS to remove residual PFA, and then treated with 0.1% Triton X-100 for 5 min following washing with PBS wash three times. Cells were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated with the following different primary antibodies overnight at 4 °C: anti-Renin (1:100 dilution, #ab212197, Abcam), anti-Piezo1 (1:100 dilution, #APC-087-25, Alomone), anti-PMCA (1:100 dilution, #MA3-914, Sigma-Aldrich), and anti-αSMA (1:200 dilution, #ab5694, Abcam). Samples were washed with 0.1% PBST buffer (PBS + 0.1% Tween 20) three times for 10 min each and incubated with fluorescently conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (Thermo Fisher) for 2 h at room temperature before being washed with 0.1% PBST buffer another three times. The nuclei were counterstained with 4′6′-diamidino-2 phenylindole (DAPI) at room temperature for 5 min, and the cells were finally washed with PBST three times. Fluorescent images were captured using an Olympus IX73 inverted fluorescence microscope or a Zeiss LSM 710 confocal microscope.

Renin protein expression was assessed by immunohistochemistry. Paraffin blocks were cut at 4 µm, deparaffinized in xylene, and rehydrated through graded alcohols. After deparaffinization, endogenous peroxidases were blocked in an aqueous solution containing 3% H₂O₂ and 10% methanol. Antigen retrieval was performed boiling the samples in citrate buffer (10 mM citric acid pH 6.0). The sections were then blocked in bovine serum albumin (5%) and incubated during 16 h at 4 °C with anti-renin primary antibodies (Dilution 1:2000, ab212197, Abcam, Cambridge, UK). Biotinylated antibodies against rabbit IgGs (Dilution 1:250, BA-9500, Vector Laboratories, Burlingame, CA, USA) were employed as secondary antibodies. Proteins were visualized using the Avidin-Biotin Complex (ABC) Peroxidase Standard Staining Kit (32020, ThermoFisher Scientific, Waltham, MA, USA) followed by 3,3′-Diaminobenzidine (DAB) Enhanced Liquid Substrate System (D3939, MilliporeSigma, Darmstadt, Germany). Counterstaining was done with Haematoxylin Gill Nº3 solution. Once stained, 10 representative images of the juxtameglomerular apparatus per section were taken at 400× magnification to measure renin stained area.