Safranin o staining kit

Samples were collected from the rats and IHC was performed as reported previously (24 ). Briefly, cartilage samples of the tibial plateau containing 2-cm thick subchondral bone were collected and sliced following 4% paraformaldehyde fixation for 4 h at room temperature, 10% EDTA (pH 7.3) decalcification, dehydration and paraffin embedding. For Safranin-O staining, the slices (5-µm thickness) were stained with 1% Safranin-O for 1 min at room temperature using a Safranin-O staining kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. The expression of MMP-13/ADAMTS5 proteins were detected by IHC staining as previously described (29 (link)). Briefly, slices were incubated with primary antibodies anti-MMP-13 (cat. no. ab39012; dilution 1:200), anti-ADAMTS5 (cat. no. ab231595; dilution 1:200; both Abcam) at 4°C overnight, and secondary antibodies at 37°C for 30 min consecutively. The samples were then incubated with streptavidin horseradish peroxidase, stained with 3, 3-diaminobenzidine for 1 min at room temperature, counterstained with hematoxylin for 30 sec at room temperature, dehydrated in a graded ethanol series (absolute ethyl alcohol, 95% ethanol and 85% ethanol, for 3 min each), and finally mounted.