P1010

Tissues were cut into sections 5 μm thick, dewaxed with xylene, and the slides were washed in decreasing concentrations of ethanol (100%, 100%, 95%, and 75%) for 3 minutes each time. Immerse slides were washed in 4% paraformaldehyde in PBS for 5 minutes. They were washed 3 times for 5 min each in PBS (p1010, Solarbio, CHN). We used 0.01 mol/L citric acid antigen repair solution (22F00120, Dingguo, CHN) and thermal repair at 98 °C for 15 min to expose the antigen epitopes. It was treated for 12 min at room temperature with 3% H₂O₂ in PBS to block endogenous peroxidase activity. It was washed 3 times for 5 min each in PBS. For the primary antibody labeling, the slides were incubated with diluted rabbit polyclonal antibodies to PLAC8, Ki-67, β-Catenin, and PCNA at 4 °C overnight. The next day at room temperature for 1 hour, it was washed 3 times for 5 min each in PBS. Tissue sections were soaked with EnVision™ + System/HRP (DAB) (GK500710, Gene Tech, CHN). The tissues were then counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting. Brown staining in the nucleus was considered an indicator of protein expression and counted using a microscope (Olympus, JPN) at a magnification of ×200. For this purpose, five fields per slide were randomly selected by the viewer for evaluation.

Mice were killed using CO₂ at a 30% chamber replacement rate. After the mice had been completely euthanized, their eyes were removed. The cornea was removed after the eyes were soaked in 4% paraformaldehyde (PFA) for 20 min. After 1.5 h in PFA, the eye cups were put in 30% sucrose overnight at 4 °C. After removing the lens, the eye cups were air dried and imbedded with ideal cutting temperature compound, 4 °C freezing for 30 min, and stored at 20 °C until sectioned at 12 μm thickness.
The retinal slices were dried at room temperature for about 30 min before being washed three times (10 min each) with 1 × PBS (Solarbio, P1010), permeabilized for 10 min at room temperature with 0.3% Triton X-100 in 1 × PBS, and incubated with blocking solution (5% bovine serum albumin in 0.3% Triton) for 1 h at 37 °C. Primary antibodies were treated with the retinal slices overnight at 4 °C. The slices were then rinsed three times (10 min each) with 1 × PBS at room temperature before being incubated with secondary antibodies for 2 h at 37 °C. Sections were stained with DAPI for 7 min and cleaned as previously described. Finally, for sealing, an anti-fluorescence quenching agent was utilized. The working dilution ratio and sources of primary and secondary antibodies are listed in Additional file 1: Table S1.

MDCK cells were seeded in 12-well plates at a density of 2.0 × 10⁵ cells/well and incubated at 37 °C with 5% CO₂ overnight. The next day, different concentrations of coptisine and A/PR/8/34 (H1N1) (MOI = 0.5) were mixed at 4 °C for 30 min before being added to the MDCK cells at 37 °C for 2 h. After that, the supernatant was removed, and the cells were washed twice with PBS (Solarbio, P1010, Beijing, China). Different concentrations of chrysin were added to the cells, which were then cultured for 24 h at 37 °C. The cytopathic effect was observed under a microscope.

We collected 1 × 10⁶ transfected MCF-7 and MDA-MB-231 cells, washed with phosphate buffered saline (P1010; Solarbio Lifesciences, China) and suspended using 1 mL of binding buffer (1×), followed by centrifugation at 300 × g in a 5810 Centrifuge. The supernatant was abandoned subsequently. All cells were then resuspended with 1 mL of binding buffer (1×), and the density was adjusted to 1 × 10⁶ cells/mL. A volume of 100 μL of cells suspension was added into each tube, and 5 μL of Annexin V-FITC was also added. A gentle blend was performed at room temperature (RT) for 5 min avoiding light, after which 5 μL of propidium iodide (PI) was added into the tube and all cells were additionally incubated at RT for 5 min in the dark. The apoptosis was detected using an Annexin V-FITC/PI apoptosis detection kit (CA1020, Solarbio Lifesciences, China) using a S1000EON flow cytometer (Stratedigm, Inc., San Jose, CA, USA), and all data were analyzed using Kaluza C Analysis Software 1.12 (Beckman Coulter, Indianapolis, IN, USA).

The brain tissue Evans blue (EB) dye extravasation method was applied to determine the extent of blood-brain barrier (BBB) damage on the 3rd day after ICH. EB (E8010, Solarbio, Beijing, China) with a final concentration of 2% was administered intraperitoneally (4 ml/kg) in anesthetized mice and allowed to circulate in vivo for 3 h (Manaenko et al., 2011 (link)). After transcardiac perfusion with phosphate buffer saline (PBS, P1010, Solarbio, Beijing, China), the right hemisphere was collected and homogenized in a glass homogenizer with PBS (1,100 μL). The brain tissue homogenates were then sonicated and centrifuged (30 min, 15,000 g, 4°C). The resulting supernatant was collected and mixed with an equal amount of 50% trichloroacetic acid (T104261, Aladdin, Shanghai, China). After incubation overnight at 4°C, the mixture was centrifuged at 15,000 g for 30 min at 4°C. Finally, the absorbance of the supernatant at 610 nm was determined using a microplate reader. The extent of BBB damage in each group (vehicle, ATO, DXM, and ATO-DXM) was evaluated by comparing the experimental group to the OD₆₁₀ of the sham group.

1000 cells per well were plated onto 6-well plates (each well contained 2 mL medium supplemented with 10% FBS). After adherence for 24 h, the cells were treated with 0 μM, 40 μM, and 80 μM liensinine for 24 h; following which, the medium was replaced with the complete medium. The cells were cultured for 14 days; subsequently, they were fixed with 4% ice-cold paraformaldehyde, stained with 0.1% crystal violet, and washed thrice with PBS (P1010, Solarbio, Beijing, China). Images of cell colonies were captured by a camera (Alpha 7R IV, SONY, Tokyo, Japan), and the cell colony numbers were analyzed using the Image J software.