40fl axioskop

The OCP samples were sterilized by Co60 gamma radiation. Cells were directly seeded onto the culture plates at a density of 2 × 10⁴ cells/cm². The culture media were refreshed with OCP-particles-containing media (1 mg/mL) after cell adhesion and this moment was set as the initial culture time. After cultured for 24 h, the cells were rinsed with PBS thrice and fixed with 4 vol% formaldehyde solution for 4 h. The fixed cells were dehydrated with a graded series of ethanol (30, 50, 60, 70, 80, 90, 95 and 100 vol%) and air-dried at room temperature for 24 h. The attachment and morphology of cells were observed by the FESEM.
Cells were cultured with OCP particles at a density of 5 × 10³ cells/cm². After cultured for 3 days, the cells were rinsed with PBS thrice and double-stained with Calcein-AM (Biotium, USA) and propidium iodide (Biotium, USA) following the instructions. The fluorescence images of cells were acquired by an inverted fluorescence microscope (Eclipsc Ti–U, Nikon, Japan) equipped with a digital camera (40FL Axioskop, Zeiss, Germany). Quantitative cell viability was measured by a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. After cultured with OCP particles for 1, 3, and 5 days, the cells were incubated with the CCK-8 working solution at 37 °C for 1 h. Afterward, the absorbance of the supernatants was read at 450 nm by the microplate reader.

Immunohistochemistry of tissue sections with rabbit anti-rat CD31 antibody (Santa Cruz Biotechnology, USA) was performed at 7 and 14 days to detect angiogenesis in the wound after intervention. Sections were deparaffinized, rehydrated, heated in a microwave oven twice for antigen recovery, treated with 3% H₂O₂ and then incubated with 5% goat serum albumin. Then, the sections were incubated overnight at 4°C with primary rabbit anti-rat CD31 antibody (1:75, Santa Cruz Biotechnology), followed by a 1-h incubation with HRP-conjugated goat anti-rabbit secondary antibody (1:200, Abcam) and visualization with a DAB kit (ZSGB-BIO, China). After being counterstained with hematoxylin, the slides were assessed with a fluorescence microscope (40FL Axioskop, Zeiss).
The three regions with the most neovascularization at low magnification (5X) on each slide were selected. Then, three randomly selected areas in each region were imaged at 20X magnification. These images were analyzed with Image-Pro Plus software. The mean number of blood vessels per image was defined as the microvascular density (MVD).