Eclipse e800 microscopy

The primary BMMs were isolated, seeded on collagen-coated plates in the density of 2.5^∗10³ cell/cm² and cultured in osteoclastogenic medium for 4 days until mature osteoclasts were formed. Then mature osteoclasts were digested and seeded on dentin slice in 96 wells (1000 cells/well). Subsequently, the osteoclasts were continued cultured in osteoclastogenic medium in the presence of indicated dosages of boldine. After 48 h, the dentin slices were obtained and wiped with q-tips to remove cells, pit lection staining was performed. The brown area on the dentin slice was recognized as the bone resorption area during the 48 h. The representative pictures were taken using Nikon Eclipse E800 microscopy (Nikon), and the resorption area was measured using ImageJ software.

All the right tibias were dissected, fixed in 70% ethanol for 3 days. After fixation, tibias were infiltrated with a mixture of 85% methyl methacrylate (Sigma-Aldrich), 15% dibutyl phthalate (Sigma-Aldrich), and 0.15% benzoyl peroxide (PolyScience) at 4°C for 9 days. Tibias were then embedded in a mixture of 85% methyl methacrylate, 15% dibutyl phthalate, and 3% benzoyl peroxide, polymerized at 37°C. Standard undecalcified sections (4 μm) were cut along the coronal plane from anterior to posterior using a Reichert-Jung microtome (Cambridge Scientific). Subsequently, von Kossa staining, Toluidine Blue and TRAP staining were performed, quantitative bone histomorphometric measurements were analyzed using the OsteoMeasure system (OsteoMetrics). The representative pictures of TRAP staining and labeling from the plastic sections were taken using Nikon Eclipse E800 microscopy (Nikon) at 400× magnification.

The d-imHCs were pretreated with 10–30 µM CCM, inoculated with HBVcc at MOI 100 in Williams’ Media E, 4% PEG for 18 h, washed and maintained in complete medium for 7 days, fixed with 4% (w/v) paraformaldehyde and permeabilized with 0.2% (v/v) Triton-X100 in PBS, incubated with anti-HBcAg antibody (ab8637, Abcam, UK) at 4 °C overnight, incubated with Alexa Fluor 568-conjugated goat-anti-mouse IgG (A11004, Life Technologies, USA) for 1 h, counterstained with DAPI (Thermo Fisher Scientific, USA), and visualized under Nikon Eclipse E800 microscopy (Nikon, Japan).