Horseradish peroxidase

IHC staining was performed on fixed tissue sections using avidin–biotin
peroxidase technique. Briefly, unstained sections were deparaffinized with
xylene and rehydrated with decreasing concentrations of ethanol. Nonspecific
binding was blocked with 4% BSA. Avidin and biotin binding sites contained in
tissue samples were blocked using a commercial avidin–biotin blocking kit
(Vector Laboratories, Burlingame, CA, USA). Sections were then incubated for 30
min at room temperature with following antibodies diluted in phosphate buffered
saline containing BSA and incubated at 4°C for overnight. Sections were washed
in ice-cold saline and incubated with a secondary biotinylated anti-mouse
immunoglobulin G. The activity of endogenous peroxidase was blocked using 0.3%
H₂O₂ in horseradish peroxidase (Vector Laboratories).
Peroxidase activity was visualized using diaminobenzidine (Vector Laboratories).
This technique uses unlabeled primary antibody, biotinylated secondary antibody,
and a preformed avidin and biotinylated horseradish peroxidase macromolecular
complex. The slides were further rinsed in water and lightly counterstained with
hematoxylin. Besides, knee joint tissue sections were also stained with Col II
and alcian blue to determine the content of cartilage and PG, respectively, in
the ECM of knee joint, and OA grade was assessed using scoring system as
described previously^{24 (link)}.

Standard histology and immunostaining protocols (including H&E and Trichome) were performed. Briefly, immunohistochemical analysis was performed on 5- to 10-μm-thick sections of mouse skin. The following primary antibodies were used: mouse monoclonal anti-Ki-67 (1:100; Novocastra), rabbit polyclonal anti-GFP (1:200; Abcam), rabbit polyclonal anti-Sdf1 (1:100; Abcam), mouse monoclonal anti-α smooth muscle actin (1:100; Abcam), rabbit polyclonal anti-CD31 (1:100; Abcam), rat anti-mouse CD45 (1:100; BioLegend), and rabbit polyclonal anti-Cxcr4 (1:100; Abcam). Immune complexes were detected with secondary antibodies conjugated with either Cy3, Cy5, fluorescein isothiocyanate (Jackson ImmunoResearch), or horseradish peroxidase (Vector Laboratories). In situ hybridization on Cxcl12 was performed per the manufacturer's instructions (Advanced Cell Diagnostics); a positive control probe was directed against RNA polymerase, and a negative control probe was directed against bacterial DapB. After staining, images were directly analyzed on an AxioM1 microscope equipped with a CCD digital camera (Carl Zeiss). The number of Ki-67⁺ cells was counted for every 250 μm of cartilage, measured at the distal tip, for each sample. A minimum of four different samples was used for each time point and then averaged.