Phalloidin

For primary cilia imaging and analysis, MLO‐Y4 cells cultured on collagen I‐coated glass‐bottom 24‐well plates (Mattek) were fixed in 10% formalin and treated with anti‐acetylated α‐tubulin primary antibody, 1:1, from a C3B9 hybridoma cell line (Sigma–Aldrich). Cilia were visualized with Alexa‐Fluor 488 secondary antibody, 1:1000 (Life Technologies) and imaged with a 100× oil objective on a Leica DMi8 epifluorescence microscope. Nuclei were stained with DAPI (Life Technologies), and F‐actin was stained with phalloidin (Santa Cruz Biotech). Cell area, and circularity, and cilia incidence and length were analyzed using ImageJ software.

Cell spreading was evaluated by using cytofluorescent staining. After
three washings in PBS, samples were fixed in 4% (w/v) paraformaldehyde
for 60 min at room temperature. Following three more washings in PBS,
samples were incubated within a working dye solution prepared with
1 μL/ml phalloidin (Santa Cruz Technology, USA) and 4′,6-diamidine-2′-phenylindoledihydrochloride
(DAPI, 1 mg/mL, Sigma-Aldrich, Ireland), to highlight filamentous
actin (f-actin) of the cell cytoskeleton and cell nuclei, respectively.
Micrographs were obtained using a Leica SP8 scanning confocal microscope
(Leica Microsystems, Germany), and image processing for nuclei orientation
was performed with custom-made scripts in Matlab.

For the immunocytochemical staining the following antibodies were used: rabbit polyclonal anti-OR10H1 antibody (OriGene, Herford, Germany; dilution: 1:100), rabbit polyclonal anti-CadherinT antibody (Cell Signaling, Leiden, Netherlands; dilution: 1:50), mouse monoclonal α-E-cadherin (Cell Signaling, Cambridge, United Kingdom, 1:100), mouse monoclonal α-Rhodopsin 4D2 antibody (AR441, Dako, Glostrup, Denmark; dilution: 1:100). For staining of β-actin Phalloidin was used (Santa Cruz, Heidelberg, Germany; dilution: 1:100). The immuncytochemical staining of the transfected Hana3A and BFTC905 cells was performed as previously described (Weber et al., 2017 (link)).
The immunohistochemical staining of human bladder cancer and normal bladder tissues was conducted using a Ventana BenchMark Ultra instrument (Ventana Medical System, Tucson, AZ, United States) and the UltraView Universal DAB detection Kit according to the manufacturer’s instructions. The primary antibody OR10H1 was diluted in antibody diluent (1:20) and applied for 32 min. All sections were counter-stained with haematoxylin (HE). An Olympus BX 43 microscope was used for visualization.

For the assessment of mitochondrial morphology in living cells, the mitochondria were stained with MitoTracker red (Life Technologies, USA) and phalloidin (Santa Cruz, USA) for 30 min at 37 °C in a humidified chamber with 5% CO₂. Images were taken using an Olympus FV10i confocal microscope. To observe the individual mitochondria, z-stack images were acquired in series of six slices per cell ranging in thickness from 0.5 to 0.8 μm per slice.

The reagenes used in current study include: PAN (Sigma, Chemical Co., St. Louis, MO, USA), SalB, RA (Shanghai Source Leaf, China), Tacrolimus (Astellas, Japan), fetal bovine serum (ExCell Biology, Shanghai, China), RPMI 1640 culture medium, penicillin and streptomycin solution, 0.5% Trypsin-EDTA (Gibco, USA), Phalloidin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), RIPA Lysis buffer, β-actin antibody, protein buffer (Beyotime Biotechnology, Shanghai, China), NF-kB, TLR2 antibody (Cell Signaling Technology, Danvers, MA, USA), TLR4 antibody (Dr. de Wuhan, China), MyD88 antibody (Boosen, Beijing, China), phospho-NF-kB (pp65) antibody (Bioworld, China) and reverse transcription and PCR kit (Tiangen, Beijing, China).

Fluorescence microscopy was used to evaluate the changes in cell structure and actin cytoskeleton. After treatment, the podocytes were washed three times with PBS prewarmed at 37 °C, and fixed with 4% paraformaldehyde for 10 min. The cells were then rinsed with PBS for three times, followed by permeabilization with 0.5% Triton X-100 for 5 min. The cells were then washed with PBS and incubated with Phalloidin (1:400, Santa Cruz Biotechnology) for 30 min in the dark at room temperature. Images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan).