Tissue slides collection
The tissues of 12 patients with ICH and the matched normal tissue slides were collected. Meanwhile, pathological characteristics of these samples were obtained, including age, smoking history, drinking history, diabetes history, systolic blood pressure, diastolic blood pressure, fasting blood glucose, and lipid levels. All patients in this study signed informed consents.
Immunohistochemical (IHC) staining
Firstly, the tissue slides were placed at 65℃ for 30 min, then dewaxed with xylene and washed with alcohol. Then the tissue slides were repaired by citrate buffer, cooled to room temperature and soaked in 1×PBST buffer (1×PBS + 0.1% Tween 20) for 5 min. Next, the tissue slides were sealed with 3% H2O2 and 5% serum for 15 min, respectively. Following, they were incubated with anti-ITGB1 antibody (1:200, abcam, Cat # ab8991) and anti-ITGB3 antibody (1:200, abcam, Cat # ab119992) overnight at 4℃, respectively. The slides were washed with 1×PBST buffer solution for 5 min/3 times, and then the secondary antibody HRP Goat Anti-Rabbit IgG (1:200, abcam, Cat #ab111909) was added and placed at 37℃ for 1h. After washing the slides at the end of the secondary antibody reaction with 1×PBST buffer, the slides were dyed for 5 min with DAB solution, and then staining was terminated by washing with H2O. After that, it was re-dyed with hematoxylin for 15 s, and finally sealed with neutral gum.
IHC scores were determined by staining percentage scores (classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%), 4 (75%-100%)) and staining intensity scores (scored as 0: signal less color, 1: brown, 2: light yellow, 3: dark brown). To distinguish between high and low expression, the median was selected as cut off-value to reduce the impact of outliers. All tissue microarray chips were pictured with microscopic and viewed with Image Scope and Case Viewer.
Animal model construction
In this study, 12 clean grade Sprague-Dawley (SD) rats (250-3008, 8-10 weeks old), half male and half female, were reared in SPF animal room. The animals were kept in cage with alternating light and dark for 12 h, keeping the feeding temperature at 20°C and humidity at 50-60%. The experiment was divided into two parts: in the first part, 12 rats were numbered one by one and randomly divided into sham operation group (n = 3) and cerebral hemorrhage group (n = 9). The animal model of cerebral hemorrhage group was injected with collagenase (0.2 U/ulVII collagenase) prepared by 2.5 μL normal saline, and the sham operation group was injected with 2.5 μL normal saline. The rats were killed at 4 days, 7 days and 21 days after the establishment of the model, respectively. 3 rats were randomly selected at each time point, and one in the control group was killed. Then, western blotting (WB) was used to detect the expression of ITGB1 and ITGB3 in the brain tissue of rats with hemorrhagic stroke.
Western blotting (WB) analysis
The experiment was divided into control (CON) and ICH group. The rats in the ICH group were sacrificed 4 days, 7 days and 21 days after modeling, and the expressions of ITGB1 and ITGB3 in the ICH tissues of the rats with hemorrhagic stroke were detected by WB. Firstly, the proteins were extracted with cell lysate, and detected by BCA protein detection kit (HyClone-Pierce). The 10-µg protein was separated by SDS-PAGE (Invitrogen) and transferred to the PVDF membrane, then sealed at room temperature for 1 h with TBST solution. After that, the membrane was first incubated with primary antibodies (ITGB1, 1:1000, abcam, Cat # ab8991; ITGB3, abcam, Cat # 1:1000, ab119992; GAPDH, 1:3000, Bioworld, AP0063) at 37℃ for 2 h. Following, the membrane was incubated with HRP-conjugated (goat anti-rabbit, 1:3000, Beyotime,Cat # A0208;goat anti-mouse, 1:3000, Beyotime, Cat # A0216) at room temperature for 1 h. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Cat # RPN2232) was used for color rendering and Chemiluminescent imager (GE, Cat # AI600) observation.