A fluorescence microscope

Cells were lysed with cold RIPA buffer and whole cell lysates were subjected to SDS-PAGE as previously reported [17 (link)]. To perform immunofluorescence staining, the cells were fixed and incubated with Alexa 488-conjugated α-Tubulin antibody. DNA was stained with Hoechst 33342 in PBS. Images were analyzed on a fluorescence microscope (Nikon Instruments Inc.).

DNA damage was assessed by TUNEL assay as described by Xu et al. (2017) [43 (link)]. Briefly, NHDF and B16F10 cells were exposed to UVB (30 mJ/cm²) and then treated with PL (1.0 mg/mL) for 24 h. The treatment concentration of PL here was identical with that found in the MTT Assay. Thereafter, NHDF and B16F10 cells were washed with PBS and fixed with 4% paraformaldehyde for 25 min, washed twice with PBS and then incubated with 0.2% Triton X-100 for 5 min. Subsequently, the cells were incubated with TUNEL reaction mixture for 1 h at 37 °C in the dark, and then stained with DAPI (5 μg/mL) solution for 10 min at room temperature. After being washed three times with PBS, the cells were visualized using a fluorescence microscope (NiKon, Tokyo, Japan).

Podocytes were fixed with 4% formaldehyde in phosphate‐buffered saline (PBS) for 10 min, and the cells were stained with Hoechst 33258 for 1 hr. Apoptotic cells were identified by the condensation and fragmentation of their nuclei, and images were captured using a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Immunohistochemical analysis was performed as described previously [13 (link)]. Immunocytochemistry analysis of MECs, MaSCs, and MaCFCs were performed following the manufacturer's instructions of Immunodetection Kit (Vector, Burlingame, CA). A fluorescence microscope (Nikon, Tokyo, Japan) was used for detecting immunofluorescence.

Cell viability was assessed by double‐staining assay using calcium fluorescein‐AM/PI. PC12 cells were seeded in a 24‐well plate and allowed to attach for 24 hrs. Then, BV‐2 cells were pre‐treated with or without 200 μM Sal for 24 hrs before being stimulated with LPS and placed in the wells containing the PC12 cells for 24 hrs. Then, the PC12 cells washed gently with PBS two times, after which 2 μM calcein AM and 15 μg·M⁻¹ PI were added to the wells, and the culture plates were incubated at 37°C for 30 min. Finally, the dye solution was removed, and the samples were washed with PBS three times. A fluorescence microscope (Nikon) was used to assess the slides.

The cells were washed with PBS, resuspended in DMEM without FBS, and plated into the inner Transwell chamber (Corning, USA). The 24-well plates were supplemented with DMEM supplemented with 10% FBS and kept in an incubator with 5% CO₂ at 37°C overnight. The cells on the inner face of the Transwell chamber were removed. The cells on the upper face of the Transwell chamber were stained with 1% purple crystal and recorded with a fluorescence microscope (Nikon).