For histological analysis, harvested mouse joints were fixed with 4% PFA for 2 days and embedded in paraffin after decalcification for 14–21 days. Sections (5 μm) were stained with fast green FCF (0.02%) and safranin O (0.1%) according to the manufacturer’s instructions.
Immunohistochemical staining was performed using the DAB staining method as previously described, with some modifications.33 (link),34 (link),102 (link) First, slides were deparaffinized and rehydrated using xylene and different concentrations of alcohol. Antigen retrieval was performed using Trypsin (ZLI-9010, ZSGB-BIO) digestion for 20 min at room temperature. Hydrogen peroxide (3%) was used to block endogenous peroxidase activity by incubating for 10 min at room temperature. Slides were then blocked with 10% donkey serum for 1 h at room temperature and incubated with an anti-P16 antibody (Ab54210, Abcam, 1:200) at 4 °C overnight and with a secondary antibody (PV-6002, ZSGB-BIO) for 1 h at room temperature before DAB staining (ZLI-9017, ZSGB-BIO).
Immunohistochemical staining was performed using the DAB staining method as previously described, with some modifications.33 (link),34 (link),102 (link) First, slides were deparaffinized and rehydrated using xylene and different concentrations of alcohol. Antigen retrieval was performed using Trypsin (ZLI-9010, ZSGB-BIO) digestion for 20 min at room temperature. Hydrogen peroxide (3%) was used to block endogenous peroxidase activity by incubating for 10 min at room temperature. Slides were then blocked with 10% donkey serum for 1 h at room temperature and incubated with an anti-P16 antibody (Ab54210, Abcam, 1:200) at 4 °C overnight and with a secondary antibody (PV-6002, ZSGB-BIO) for 1 h at room temperature before DAB staining (ZLI-9017, ZSGB-BIO).
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