4 6 diamidino 2 phenylindole dapi

Mice were transcardially perfused with normal saline, followed by 4% paraformaldehyde solution for 10 min at 14 days after MCAO. Brains were fixed overnight at 4 °C, soaked in 30% sucrose solution, frozen, and cut into 10-μm-thick sections (Leica, Wetzlar, Germany). BrdU/von Willebrand factor (vWF), VEGF/vWF, VEGFR2/vWF, Ang-1/vWF, and Tie-2/vWF were detected by double immunofluorescence staining as described in our previous study [23 (link)]. Cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Zsbio, Beijing, China) at room temperature.

After treatment for 72 h, the mice were sacrificed, and the eyeballs (n = 3) were enucleated and subsequently fixed in 4% immunohistochemical fixation solution for 2 h. After rinsing with phosphate buffer solution (PBS) three times, all of the corneas were clipped along the margin of the keratosclera and then incubated with anti-β3-tubulin (1:500, ab52623, Abcam, Cambridge, United Kingdom), anti-SP (1:400, ab10353, Abcam) and anti-CGRP (1:400, 14959s, Cell Signaling Technology, United Kingdom) for 48 h at room temperature. After washing with PBS, the cornea was incubated with Alexa Fluor® 488 goat anti-guinea pig IgG H&L (1:500, ab10353, Abcam) and Alexa Fluor® 594 goat anti-rabbit IgG H&L (1:400, ZF-0516, ZSGB-BIO, China) at 4 °C overnight. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI, ZSGB-BIO, China) to stain the nuclei, all of the images were captured by using a fluorescence microscope (Nikon, Tokyo, Japan). To compare the changes in corneal innervation, Image J and Neuron J software were used to calculate the nerve length in the positive area for β3-tubulin staining per sample. In the experiment, one representative image in the center of the cornea and four replicates captured in the peripheral area of cornea from three independent experiments per group were used for analysis.

Immunofluorescence staining was performed on well-cultured fibroblasts. In brief, after fixation, serum blocking and hybridization with primary antibodies overnight, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100 dilution; KeyGEN, Nanjing, China) for 1 h, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; ZSGB-BIO, Beijing, China). The cells treated with only secondary antibodies were considered the negative control (NC). The fluorescent expression of the target markers and nuclei were evaluated and imaged using a Leica confocal laser microscope.

Cells at a density of 1 × 10⁵ cells/well were seeded into 12-well plates. After exposure to the THP treatments, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Then, the cells were made transparent with 1% PBST for 10 min at 4℃. After the PBST was aspirated, the cells were blocked with 4% bovine serum albumin (BAS) for 30 min and then incubated with a primary antibody against p-P38 (1:200, ImmunoWay, USA), iNOS (1:200, Proteintech, USA), and GFAP (1:200, CST, USA), in antibody dilution buffer overnight at 4 °C. The next day, the cells were incubated with a fluorochrome-conjugated anti-rabbit secondary antibody (1:200, ZSGB-BIO, China) for 2 h at room temperature in the dark. Subsequently, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml, ZSGB-BIO, China) for 15 min. Images were obtained under a fluorescence microscope. Quantitative analysis of the immunofluorescent staining was performed using Image J.

Serial sections in thickness of 4 μm were cut through the paraffin-embedded left lung lobe and stained with HE (Beijing Dingguo Changsheng Biotechnology, China). Anti-α-SMA (1:200, Sigma-Aldrich, USA) and FSTL1 (1:200, Santa Cruz, USA) were visualized by Alexa Fluor 594-labelled goat anti-mouse IgG and Fluorescein-conjugated rabbit anti-goat IgG (ZsBio, China), respectively, with nuclei mounted by 4′,6-diamidino-2-phenylindole (DAPI, ZsBio, China). Blood vessels were screened with a microscope digital camera (Nikon, Japan) and analyzed by NIS-Elements system (Nikon, Japan). Vascular remodelling was evaluated by MT% and numbers of completely muscularized arterioles52 (link)53 (link). Briefly, MT% was expressed as a percentage of ((external diameter - internal diameter)/external diameter). Arterioles exhibiting more than 75% of circumference positive for α-SMA were identified as completely muscularized arteries and their numbers were totaled in every 10 high-power (×400) fields. Transversely cut arterioles were included for measurement, with the exclusion of obliquely cut ones and pulmonary veins.