Ri 1 microscope

Briefly, 6-μm sections of knee joint tissue were placed on positively charged glass slides (Thermo Fisher Scientific, Asheville, NC, USA) and then dried with a hotplate to increase the adherence of the tissues to the slides. After the samples were deparaffinized and rehydrated according to conventional methods, endogenous peroxidases were blocked with 3% hydrogen peroxide in methanol (Sigma-Aldrich, Burlington, USA). After 30 min the samples were digested with 5 mg/ml hyaluronidase in phosphate buffered saline (PBS) (Sigma-Aldrich) for 20 min and then were incubated with anti-CD31 (1:100 dilution) (SC-56; Santa Cruz, CA, USA) and anti-CD34 (1:100 dilution) (Santa Cruz, CA, USA) antibodies overnight at 4 °C. Following treatment of the samples with appropriate biotinylated secondary antibodies, a 3,3’diaminobenzidine (DAB) streptavidin-peroxidase (SP) DAB Histostain-SP immunohistochemistry kit (ZYMED203 Laboratories/Invitrogen, Carlsbad, CA, USA) was used to visualize bound antibodies. After the sections were counterstained with hematoxylin (ZYMED Laboratories/Invitrogen), the tissues were imaged with a Nikon Ri 1 microscope (Nikon, Melville, NY, USA) (Guan et al. 2012 (link)).

Right knee joints were harvested from animals which were euthanized on postnatal days 14 and 21. The knee joints were fixed in 4% neutral buffered formalin for 24 h at 20 °C and then were processed without decalcification and embedded in a single block. Sections were cut from the sagittal plane, with adjacent sections collected at intervals of 0, 100, and 200 μm from the cartilage layer. Two serial 6-μm-thick sections from each interval were stained with Safranin O and then mounted on slides in order to evaluate development of the growth plate and secondary ossification center. Thickness of the growth plate and ratio of the area of secondary ossification center to the area of tibial plateau were subsequently quantitated from images collected with a Nikon Ri 1 microscope (Nikon, Melville, NY, USA).