Goat anti rabbit immunoglobulin g igg

After 48 hours of infection, GSCs were collected and lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). A Bicinchoninic acid kit (Pierce, USA) was used to detect the protein concentration in the supernatant. Proteins were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membrane was blocked with 5% skim milk and probed with rabbit antibodies against jagged canonical Notch ligand 1 (JAG1; ab7771, 1:500), NOTCH1 (ab8925, 1:500), Presenilin‐1 (PS1; ab76083, 1:5000), HES family bHLH transcription factor 1 (HES1; ab71559, 1:1000) or GAPDH (ab9485, 1:2500, internal reference). All of aforementioned antibodies were obtained from Abcam (Cambridge, UK). The membrane was then re‐probed with goat anti‐rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) conjugated by horseradish peroxidase (HRP) for 1 hour. Enhanced chemiluminescence (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to develop the membrane, which was visualized in a Bio‐Rad ChemiDoc™ image analysis system. Protein bands were analysed by ImageJ2x software.

Random samples from femoral heads of various OA stages were immunostained with the two-step immunohistochemistry method as instructed by the manufacturer. In brief, deparaffinized sections were incubated overnight with rabbit polyclonal antibody against lubricin (sc-98454, Santa Cruz Biotechnology INC., CA, USA), 1∶200 dilution, at 4°C. The slides were washed three times in PBS followed by 30 min incubation at room temperature with goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology INC., CA, USA) and visualized with DAB chromagen. Stained sections were photographed with a Ziess AxioCam digital camera, at 20× magnification with constant illumination intensity (Figure 2).