Ab84956

After deparaffinization, the slices were rehydrated; washed with PBS; incubated with 3% hydrogen peroxide at room temperature for 15 minutes; washed with PBS; incubated with hyaluronidase IV at 37 °C for 30 minutes; washed with PBS; blocked with 1:10 normal blocking serum at room temperature for 45 minutes; and incubated with a 1:100 dilution of anti-COL-I (ab84956, Abcam), a 1:100 dilution of anti-Osterix (ab22552, Abcam) or a 1:100 dilution of anti-COL-X (ab58632, Abcam) at 4 °C overnight. The slides were washed and incubated with a secondary antibody (sp9000, ZSGB-BIO) at 37 °C for 20 minutes, washed with PBS incubated with streptavidin peroxidase at 37 °C for 20 minutes. Then, the slides were washed with PBS and exposed for 5 minutes to the peroxidase DAB substrate (ZSGB-BIO, China). After staining with hematoxylin, the slides were examined with an Olympus microscope mounted with an Olympus video camera. We used Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) to analyze the results. We first circled the area of interest and then measured the cumulative optical density (OD) of what we stained. Afterward, we determined the average OD with the cumulative OD divided by the size of the area of interest.