Acc 017

These methods were described previously [13 (link)]. Renal cortex was homogenized in M-PER™ mammalian protein extraction buffer (Thermo Fisher Scientific) and sonicated on ice. The homogenates were subjected to centrifugation at 13,000 rpm for 30 min. Protein concentrations of the supernatants were determined with Bradford reagent (Bio-Rad). Rabbit antibodies against TRPC6 (ACC-017) and TRPC3 (ACC-016) were from Alomone Labs. The TRPC6 antibody targets motifs encoded by exon 1 located in the amino-terminal and upstream of the deletion in the Trpc6^del allele. We also used a polyclonal TRPC6 antibody from Booster Biological Technology that targets residues 248–264 in the rat channel, downstream of the deletion. A mouse monoclonal antibody against TRPC5 was from NeuroMAB. We also used a rabbit polyclonal antibody against TRPC5 from Alomone (ACC-020). All TRPC antibodies were used at a dilution of 1:1000. A mouse monoclonal antibody (clone 1A4) against α-smooth muscle actin (SMA) was from Sigma-Aldrich.

After 4 µm sections were collected, the paraffin on the sections was removed by xylene, and then hydrated in graded ethanol. The sections were incubated for 10 min in 10 mmol/L sodium citrate buffer in microwave oven for the purpose of retrieve antigenicity. After that, the sections were incubated with antibody against Col-IV (ab6586 Abcam, Cambridge, MA), TGF-β₁ (ab92486 Abcam, Cambridge, MA), and TRPC6 (ACC-017, Alomone Labs, Belmont, CA) overnight at 4 °C. The sections were washed fully by phosphate-buffered saline (PBS) solution and incubated with second antibody (Shanghai Changdao, Co., Inc., Shanghai, China) for 30 min. PBS solution was used to replace specific antibody for the purpose of obtaining negative controls. Brownish yellow granular in the glomerulus were considered as positive areas. Semi-quantitative evaluation was performed by Image-Pro Plus6.0 (NIH Image J system, Bethesda, MD).

Slides were deparaffinized, rehydrated, and heated in citrate buffer pH 6 for antigenic retrieval. After blocking for endogenous peroxidase with 3% hydrogen peroxide, the following antibodies were incubated: TRPA1 (#ACC-037 1:500, 1 h, Alomone Labs, Jerusalem, Israel), TRPV2 (SAB1101376 Sigma, 1:1000, 1 h), TRPC3 (#ACC-016 Alomone, 1:500, 1 h), TRPC6 (#ACC-017 Alomone, 1:300, 1 h). Immunohistochemistry was performed using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (Kit LSAB, Dakocytomotion, Glostrup, Denmark). Slides were finally counterstained with haematoxylin. Negative controls were obtained after omission of the primary antibody or incubation with an irrelevant antibody. For cryo-sections, retinas were fixed overnight with 4% PFA, rinsed in PBS and then incubated with 30% sucrose before OCT embedding. Retina sections were blocked and permeabilized in PBS 0.25% triton, 5% FBS, 1% BSA for 2 h and incubated overnight at 4 °C with the rabbit anti-TRPA1 antibody (#ACC-037 Alomone, 1:100) and the rat anti-CD31 (Becton Dickinson, 1/100). After washes, sections were incubated with secondary antibody donkey anti-rabbit A488 and donkey anti-Rat A594 for 2 h at room temperature. Sections were then washed and stained with DAPI before mounting in Mowiol. Sections were imaged with a Carl Zeiss AxioImager Z1-Apotome.

The cells cultured on confocal dishes were fixed with 4% formaldehyde in 0.1 M PBS for 15 min and washed three times with 0.1 M PBS. Further details were performed as previously described ³⁶6. The cells were incubated with primary specific antibodies against TRPC6 (ACC-017; Alomone Labs, ISR) at 4 °C overnight and rewarmed for 30 min subsequently. Following 3 washes with PBS, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Life Technology, USA) for another 1 h. The nuclei were stained by the fluorescent DNA-binding dye 136 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Roche, CHN) for 15 min. Fluorescence pictures were taken under a confocal laser scanning microscopy (SP8; Leica TCS, GER).

These methods were previously described in detail [18 (link)]. Rabbit antibodies against TRPC6 (ACC-017) and TRPC3 (ACC-016) were obtained from Alomone Labs (Jerusalem, Israel). A mouse monoclonal antibody against TRPC5 was obtained from NeuroMAB (Davis, CA, USA). All TRPC antibodies were used at a dilution of 1:1000. The antibody against vimentin (cloneV9) was from DAKO (Agilent, Santa Clara, CA, USA) and used at a dilution of 1:1000. The antibody against NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) was obtained from LSBio (Seattle, WA, USA), and was used at a dilution of 1:1000.