Cx31 32rfl

Sections of the brain and cultured cells were labeled with the TUNEL Bright Red Apoptosis Detection Kit System (Vazyme Biotech, USA) in accordance with the instructions of the TUNEL kit. The sections were observed by Laser Scanning Confocal Microscope (CX31-32RFL, Olympus, Tokyo, Japan). The apoptotic cells were counted by Image J software (ver. 1.61). The apoptotic ratio was calculated as apoptotic cells / total cells × 100%.

Cy3-labeled lncRNA PEG11as probes were designed and synthesized by BersinBio (Guangzhou, China). The probe signals were determined with the Fluorescent in Situ Hybridization Kit (RiboBio, Guangzhou, China) in accordance with the manufacturer's guidelines. In brief, the N2a cells grew to 60-70% con uency on the slides and were xed with 4% paraformaldehyde (PFA). Cells were pre-hybridized in 0.5% TritonX-100-PBS at room temperature. Later, the FISH probes were hybridized in a hybridization buffer in the dark at 37 °C overnight. After rinsing with the saline-sodium citrate (SSC) buffer, nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). The images were acquired using a confocal laser microscope (CX31-32RFL, Olympus, Tokyo, Japan).

Apoptosis in cells transfected with PFN1 siRNA was examined using Hoechst Staining Kit System (Beyotime, China) following the manufacturer's manual. In brief, the N2a cells were cultured in 24-well plates with 5 × 104 cells per well, then transfected with PFN1 siRNA when the cells were grown to 60-70% con uency. Forty-eight hours after transfection, the cells were treated with OGD 3h / R 24h, and then xed with 4% paraformaldehyde at 4°C for 10 min. Following, the cells were washed with PBS and stained with Hoechst 33258 solution in the dark for 10min. The N2a cells were observed using a Laser Scanning Confocal Microscope (CX31-32RFL, Olympus, Tokyo, Japan). The apoptotic ratio was then, calculated as apoptotic cells / total cells × 100%.

The removed kidneys were weighed and the cortices were then separated. The left cortices were snapfrozen for Western blotting and real-time PCR analyses, and the right cortices were used for pathological analyses. The renal tissues were fixed in Bouin's solution, embedded in paraffin, cut into 5-mm sections, and stained with periodic acid-Schiff (PAS) and H&E staining. For immunofluorescence analysis, the renal cortex of 1 mm × 1 mm × 1 mm was fixed in 2.5% glutaraldehyde and the remaining kidney tissues were fixed in 4% paraformaldehyde.
The podocytes were stimulated by 25 mmol/Lof glucose for 36h, and fixed in 4°C 4% paraformaldehyde for 20 min and 0.6% Tween-20 for 15min, and then blocked with 5% BSA blocking solution for 20 min at room temperature. The cells were exposed to antibodies against VDR, Nephrin, Podocin, α-SMA, and MMP9 overnight. The cells were washed three times with PBS, and then incubated with the Alexa Fluor 488 goat anti-rabbit IgG (H+L) (green) and the Alexa Fluor 594 donkey anti-mouse IgG (H+L) (red) (Invitrogen Inc., Carlsbad, CA, USA) at 37°C for 1 h. The fluorescence was observed under a fluorescence microscope (Olympus, CX31-32RFL, Japan).