Immunohistochemical staining

The immunohistochemical analysis was performed on paraffin-embedded tissues as previously described [55], Briefly, the deparaffined sections were incubated with 0.3% H₂O₂ in methanol to inhibit endogenous peroxidase activity, and non-specific binding was blocked by incubating sections with 5% BSA for 1 h at room temperature. The sections were probed with antibodies against renin (1:100 dilution, #ab212197, Abcam), except they were counterstained with hematoxylin if applicable. The stained sections of each group were examined of the average optical density (AOD) of renin protein. Five randomly fields of each section were used for analyzing the positive staining as previously reported [55, 56] using the ImageJ software, and the AOD values of each sample were used as an index of the expression of renin.

For multiplexed immunohistochemical staining using the Opal protocol [57, 58], briefly, the slides were deparaffinized in xylene and rehydrated in ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) using microwave heating (MWT). Primary rabbit antibodies for renin (1:100 dilution, #ab212197, Abcam) were incubated for 1 h in a humidified chamber at room temperature, followed by detection using the rabbit SuperPicture Polymer Detection HRP kit. Visualization of renin was accomplished using 1 × Opal 570 TSA Plus, after which the slide was placed in citrate buffer (pH 6.0) and heated using MWT. In a serial fashion, the slide was then incubated with primary rabbit antibodies for piezo1 (1:100 dilution, #15939-1-AP, Proteintech). Samples for 1 h in a humidified chamber at room temperature, followed by detection using the rabbit SuperPicture Polymer Detection HRP kit. Piezo1 was visualized using 1 × Opal 620 TSA Plus. The slide was again placed in citrate buffer (pH 6.0) and subject to MWT, and then incubated with primary rabbit antibodies for αSMA (1:100 dilution, #ab5694, Abcam) for 1 h in a humidified chamber at room temperature, followed by detection using the rabbit SuperPicture Polymer Detection HRP kit. αSMA was then visualized using 1 × Opal 690 TSA. The slide was again placed in citrate buffer (pH 6.0) and heated using MWT. Nuclei were subsequently visualized with DAPI (1:2000) for 5 min, and the section was coverslipped using Vectashield Hardset mounting media. Using this Opal method, three primary antibodies were sequentially applied to a single mouse kidney slide. Visualization of 4-color Opal slides can be performed using Mantra or Vectra Quantitative Pathology Imaging Systems.