4 6 diamidino 2 phenylindole dapi solution

The colon of the rat was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin, and tissue sections (5 μm) of the colon were prepared for immunofluorescence analysis. For in vitro experiments, HT-29 cells were plated in 12-well plates and treated with drug serum or PMA (1 μM; Beyotime, Shanghai, China) for 24 h. After washing with PBS twice, cells were fixed in 4% paraformaldehyde for 10 min at room temperature and prepared for immunofluorescence analysis.
For immunofluorescence analysis, tissue sections (5 μm) of the colon or HT-29 cells were permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and then incubated with primary rabbit anti-AQP3 antibody (ab125219; Abcam, CA, USA) overnight at 4°C. Then, the tissue and cells were washed twice with PBS/0.1% Tween-20 and incubated with a secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 for 1 h at room temperature. Then, the tissue and cells were reacted with 4, 6-diamidino-2-phenylindole (DAPI) solution (Beyotime) in PBS at room temperature for 5 min and then observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) and photographed. The expression level of AQP3 in rat colon or HT-29 cells was analyzed by ImageJ software.

The chondrocytes were inoculated in a 12-well plate (cell density: 2.0 × 10⁵/well), and then treated with 10⁻⁸ M 1α,25-(OH)₂D₃ and/or 2 μM Cd acetate for 72 h. After treatment, the chondrocytes were washed in PBS for 2 min on a shaker, fixed in 4% paraformaldehyde at room temperature for 30 min and subsequently washed in PBS 3 times. The chondrocytes were then treated with 0.4% Triton X-100 (Amresco, USA) for 20 min, the antigens blocked with 5% bovine serum albumin at room temperature for 30 min, incubated with anti-COL2A1 primary antibodies (Abcam, UK) overnight at 4°C, washed 3 times in PBS, and then incubated with fluorescence secondary antibodies (Cell Signaling, USA) in the dark for 1 h. The cytoskeletons of chondrocytes were stained with phalloidin solution at room temperature for 15 min. After rinsing with PBS, the chondrocytes were further incubated with 4',6-diamidino-2-phenylindole (DAPI) solution (Beyotime, China) at room temperature for 5 min nuclear staining. The glass slip in each culture plate well was removed and placed upside down on a glass slide with anti-fluorescence quenching and mounting medium, sealed with nail polish, air-dried, and stored in the dark at 4°C before observing and photographing under a laser confocal microscope (Leica).

Brown planthopper ovaries were separately collected on the 1st, 3rd, and 5th day post dsRNA injection. The DNA fragmentation in BPH ovarian cells was evaluated with the DeadEnd^TM Fluorometric TdT-mediated dUTP Nick-End Labeling (TUNEL) System (Promega) according to the manufacturer’s instructions. The fluorescein-12-dUTP provided in the kit was used to label the broken DNA in apoptotic cells. The nuclei were counterstained with a 4′,6-diamidino-2-phenylindole (DAPI) solution (Beyotime Biotechnology). Apoptotic cells labeled positively by fluorescein-12-dUTP (green fluorescence) and total cells with a DAPI staining (blue fluorescence) were imaged under a Zeiss LSM780 confocal laser scanning microscope (Zeiss, Göttingen, Germany).

By use of the BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China), EdU assay was implemented in DU145 and LNCAP as instructed by provider. After transfection, cells (1 × 10⁵ per well) were seeded into 6-well plates before being washed with PBS, and then EdU medium was added into cells for 2 h. Next, 4% paraformaldehyde (PFA) was added for 15-min fixing. Cell nucleus was visualized via 4′,6-diamidino-2-phenylindole (DAPI) solution (Beyotime) using fluorescence microscope (Olympus, Tokyo, Japan). Each independent experiment was carried out at least thrice.