Gb21404

The different groups of chondrocyte sheets were fixed in 4% paraformaldehyde and embedded in paraffin. Cell sheets were incubated with Type II Collagen primary antibodies (rabbit anti human, Servicebio, GB11021, 1:100 dilution), MMP16 (goat anti human, R&D, AF1785, 1:40 dilution) and a secondary antibody (donkey anti rabbit, Servicebio, GB22403, 1:200 dilution and donkey anti goat, Servicebio, GB21404, 1:300 dilution). The cell nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI, Servicebio, G1012). The samples were then observed and photographed observed under a high-quality fluorescence microscope.

Placental tissues fixed in 4% paraformaldehyde were paraffin-embedded and sectioned at 5 μm thickness for von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule-1 (CD31) immunofluorescence. Placentae were deparaffinized in xylene and rehydrated in grade alcohol. Placental tissues were dipped in citrate buffer (Servicebio, Wuhan, China) at 120°C in a pressure cooker for 5 min and washed 3 times in PBS (pH 7.4) for 5 min. Slides were then blocked with 3% bovine serum albumin for 30 min, followed by incubation with the vWF primary antibody (GB11020; Servicebio, Wuhan, China) and CD31 primary antibody (GB13063; Servicebio, Wuhan, China) diluted to 1 : 200 in PBS overnight at 4°C. Then, the slides were washed 3 times for 10 min in PBS (pH 7.4) followed by incubation for 30 min in a CY3 rabbit anti-goat antibody (GB21404; Servicebio, Wuhan, China) diluted to 1 : 300 in PBS. Next, slides were washed 3 times for 5 min in PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) solution in a dark room for 10 min. Finally, the slides were visualized under a fluorescent microscope (Nikon Eclipse C1, Tokyo, Japan). Fluorescence intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).