Horseradish peroxidase conjugated goat anti rabbit secondary antibody

Exosomes were lysed in lysis buffer containing a complete protease inhibitor tablet (Roche, Swiss). Five micrograms of proteins was loaded onto polyacrylamide gel, separated by electrophoresis, and then transferred to the polyvinylidene difluoride membrane (PVDF). After blocking with 5% nonfat milk, PVDF membrane was incubated with rabbit monoclonal anti-human CD63, CD9, and CD81 antibodies (Abcam, USA) overnight at 4°C, followed by the incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (BOSTER Biological Technology, China). Proteins were visualized by the enhanced chemiluminescence system (Alpha Innotech, USA).

After deparaffinization in xylene and rehydration through an ethanol series, the sections (3 μm) were washed in PBS buffer and incubated with 3% methanolic hydrogen peroxide and 10% standard goat serum. Following antigen retrieval, the slides were incubated with primary antibodies against intercellular adhesion molecule- (ICAM-) 1 (1 : 2000, Abcam, CA, USA) and P-selectin (1 : 100, Boster, Wuhan, China) at 4°C overnight. After washing with PBS, the slides were incubated in horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Boster, Wuhan, China) for 30 min at 37°C. After visualization with 3,3-diaminobenzidine tetrahydrochloride (Boster, Wuhan, China), hematoxylin was used to counterstain the sections. Finally, the images were obtained using Nikon light microscopy (Nikon Ni-U, Japan). All images were evaluated with ImageJ software (NIH, Bethesda, MD, USA).

The protein expression of Mkx and other tendon-specific genes were measured with Western blot using the same condition as described previously [26 (link)]. Proteins were extracted using RIPA Lysis Buffer (Shanghai Weiao Biotechnology Co., Ltd., China). BCA protein reagent kit (Beijing Solarbio Science & Technology Co., Ltd., China) was used to measure the concentration. Thirty micrograms proteins were run on SDS-PAGE gels (8%) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were blocked with 10% skim milk at room temperature for 1 h and were incubated with anti-Mkx (1:200, Aviva Systems Biology, San Diego, USA), anti- Col-1a1 (1:400, Bioss, Beijing, China), anti-Collagen type III (Col-3a1, 1:400, Bioss, Beijing, China), anti-Dcn (1:400, Bioss, Beijing, China), and anti-Tnmd (1:400, Bioss, Beijing, China) primary antibodies solution and anti-β-Actin (1:1000, BOSTER Biological Technology Co., Ltd, China) primary antibody solution overnight at 4 °C. After reacting with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (BOSTER Biological Technology Co., Ltd, China) for 1 h at room temperature, proteins were detected with ECL hypersensitive chemiluminescence kit (Shanghai Weiao Biotechnology Co., Ltd., China) according to the manufacturer’s recommendations.