Vs120 whole slide imager

Femoral bones were removed and fixed in 10% formalin for 24h, decalcified in EDTA-glycerol solution for 14 days, and embedded in paraffin. Specimens were cut at 5-μm and stained with for TRAP activity. In brief, sections were preincubated in Naphthol AS-BI Phosphate Substrate (Sigma) solution for 45 minutes at 37C, and incubated in Sodium Nitrite (Sigma) and Pararosaniline Dye (Sigma) solution for 15 minutes at room temperature. Sections were then incubated in phosphomolybdic acid for 5 minutes and then counterstained with 1% fast green. Sections were converted to virtual slides using an Olympus VS120 whole slide imager and osteoclast numbers were assessed using an App developed using Visiopharm software. For calcein labeling, mice were injected intraperitoneally with 10 mg/kg calcein (Sigma) per gram body weight at 7 and 1 days prior to sacrifice, as reported previously [28 (link)]. Left femora were embedded in LR white acrylic resin (Sigma), and 5-μm sections were imaged using fluorescence microscopy. The mineral apposition rates (MARs) and bone formation rates (BFRs) were calculated using a previously reported method [24 (link)].

Decalcified knee samples were used. For paraffin sectioning, 30 consecutive 4-μm-thick sections were collected and evenly divided to 3 levels. One section from each of the 3 levels was stained with Alcian blue/Hematoxylin/Orange G (AB/OG). For frozen sectioning, 10 consecutive 7-μm-thick sections were collected. Section #4 or #5 was stained with H&E, an adjacent section was subjected to double immunofluorescence staining for lymphatic vessels and macrophage subsets using primary Abs, including podoplanin (PDPN, 1:200 Abcam) for LECs, F4/80 (1:50 BioLegend) for pan macrophages, iNOS (1:50 Santa Cruz) for M1s or CD206 (1:100 R&D) for M2s. Stained sections were scanned with an Olympus VS120 whole slide imager (5 ). For 3 dimensional (3D) reconstruction, two 30-μm-thick frozen sections were stained with anti-F4/80/iNOS/PDPN or F4/80/CD206/PDPN Abs, respectively, and were scanned with an Olympus FV1000 confocal microscope to collect a series of 20–25 images. Images were imported to Amira6.0 software for 3D reconstruction.