Inverted phase fluorescence microscope

The rat kidneys were paraffin embedded, sectioned at 4 μm, and subjected to antigen retrieval followed by blocking with 1% BSA for 1 hr at room temperature to block non‐specific binding. Cultured podocytes under different conditions were plated onto different 6‐well plates and fixed in 4% paraformaldehyde for 10 min., followed by 1% BSA (with 0.3% Triton X‐100) for 1 hr at room temperature to block non‐specific binding. Immunostaining was performed with appropriate primary antibodies at 4°C overnight and Dylight 594‐conjugated IgG at room temperature for 1 hr for visualization. 4′,6‐diamidino‐2‐phenylindole (DAPI) was used to visualize the nuclei. For the staining of F‐actin, FITC‐phalloidin (50 μg/ml) was used before DAPI at room temperature for 1 hr. Images were observed and captured on an inverted phase/fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).

MALAT1 probe and fluorescence in Situ Hybridization kit were purchased from Ribio (Guangzhou, China). Briefly, cultured podocytes under different conditions were plated onto different 6‐well plates and fixed in 4% paraformaldehyde for 10 min., followed by ice‐cold PBS containing 0.5% Triton X‐100 for 5 min., and then incubated with pre‐hybridization buffer for 30 min. at 37°C to block non‐specific binding. Hybridization buffer was preheated in a 37°C water bath, and the MALAT1 FISH probe working buffer was prepared by diluting probe stock solution in hybridization buffer (1:40). Cells were incubated with probe working buffer at 37°C overnight and followed by DAPI staining to visualize the nuclei. Images were observed and captured on an inverted phase/fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).