Dp72 microscope

Transwell migration assay was performed using transwell inserts (BD bioscience, SanJose, CA) with a filter of 8 μm pore. Cells were left untreated or treated with DOX, RES or both for 48 h. 8 × 10⁴ cells in serum-free medium were seeded into the upper chamber of the insert and complete medium was added to the lower chamber. After 36 h incubation, the cells were fixed with methanol and stained with Giemsa. Then cells on the top surface of the membrane were wiped off, and cells on the lower surface were examined with Olympus DP72 microscope at 100× magnification. 4 random fields were photographed for counting purposes and the average number of migrated cells was used as a measure of migration capacity.

For the immunohistochemical studies, the sections were incubated overnight at 4 °C with the monoclonal rabbit anti-mouse primary antibody (PCNA, 1/500; Abcam, Cambridge, CA, USA) and the polyclonal cleaved caspase-3 primary antibody (1/200, Abcam, Cambridge, CA, USA). Then, the sections were rinsed with 0.01 mol/L PBS (pH 7.4) and incubated with biotinylated sheep anti–rabbit IgG (1/300; Sigma, Cambridge, CA, USA) for 2 h at room temperature. After washing, the tissues were incubated with streptavidin–horseradish peroxidase (1/300, Sigma, St. Louis, MO, USA) for 2 h and 30 min at room temperature. Immunoreactivity was visualized by incubating the tissue sections in 0.01 mol/L PBS containing 0.05% 3′,3–diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO, USA) and 0.003% hydrogen peroxide for 10 min in the dark. The sections were then stained with hematoxylin and mounted. The images were acquired on an upright DP72 microscope (Olympus, Tokyo, Japan). Image analysis system (Image-pro plus 6.0) was used to count the number of PCNA–positive cells and measure the mean integral optical density (IOD) of cleaved caspase-3 positive cells. The data were counted from 10 random fields from three cross-sections of the three specimens from each group.

For assay of cell viability and proliferation, treated cell viability was detected by CellTiter-Glo Luminescent Cell Viability Assay from Promega as described before^{35 (link),36 (link)}. GFP- LC3 assay was performed in MCF-7 cells transiently transfected with GFP-LC3 constructs, and then the cells were cultured on coverslips and then treated for the indicated times. Fluorescence was immediately observed using an Olympus DP72 microscope (Olympus Corporation, Tokyo, Japan).

H₂O₂ and superoxide anions (O²⁻) were detected in situ using 3,3ʹ-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining as previously described (Ren et al., 2019 (link)). Briefly, WT and dek66 mutant kernels isolated from self-pollinated segregating ears at 15 DAP were longitudinally cut and immersed in 1 mg ml⁻¹ DAB (Sigma-Aldrich) in 50 mM Tris–HCl buffer (pH 5.0) at room temperature in dark for 12 h to detect hydrogen peroxide (H₂O₂). NBT staining was performed to detect O²⁻. Briefly, the kernels were immersed in 0.5 mg ml⁻¹ NBT solution in 10 mM potassium phosphate buffer (pH 7.6) and incubated in complete darkness at room temperature for 20 min. The images were taken using an Olympus DP72 microscope.

Hematoxylin and Eosin (H&E) staining was performed according to a previously described method (1 (link)). Briefly, the middle portions of colon tissues were excised and immobilized in 10% (100 g/L) formalin for 24 h, followed by dehydration with gradient ethanol. Next, the colonic tissues were made transparent with xylene, embedded in paraffin, sliced into 5-μm-thick sections, stained with H&E, and observed using a DP-72 microscope (Olympus, Tokyo, Japan) to assess the morphometry of intestinal epithelial cells, neutrophil density, loss of crypt glands, and lymphocyte infiltration.