Paraformaldehyde (pfa)

After the induction of osteogenic differentiation, mineral deposition was assessed by ARS (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 15 min at room temperature and then washed with distilled water twice. A 1% solution of Alizarin Red was added and incubated for 10 min at room temperature; this was followed by rinsing with distilled water. Photographs of images were then taken using an inverted microscope (Leica, Wetzlar, Germany).
After the induction of adipogenic differentiation, fat droplet was assessed by Oil Red O staining kit (Cyagen Biosciences). Cells were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 15 min at room temperature and then washed with distilled water twice. An Oil Red O staining was added and incubated for 30 min at room temperature; this was followed by rinsing with distilled water. Photographs of images were then taken using an inverted microscope (Leica, Wetzlar, Germany).

The animals were killed under general anesthesia provided by Jiagan Biotech Company (pentobarbital; Sinopharm Chemical, China) at 30 mg/kg intravenously. The carotid arteries were exposed and cannulated. The animals were euthanized with additional pentobarbital (Sinopharm Chemical, China) at a dose of 90 mg/kg intravenously. The animals were perfused with 4% paraformaldehyde (Sangon Biotech, Shanghai, China). The involved teeth were extracted and fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 24 h at 4 °C. The samples were demineralized in 10% EDTA for 2 months at 37 °C then embedded in paraffin. The sections with a thickness of 5 μm were cut in a mesiodistal direction for HE staining and immunohistochemistry (IHC).

The expression levels of apoptosis-related genes after BmNPV-induced apoptosis in BmβGRP4 overexpressed BmN cells were detected by RT-qPCR analysis. The BmβGRP4 overexpressed BmN cells were infected with BV-EGFP at an MOI of 2 and the cells were collected at 36 h p.i. to analyze the relative expression levels of BmApaf1, BmDredd, BmBuffy, BmCaspaseNC, BmICE and BmCaspase1 through RT-qPCR; all of the primers are shown in Table S2. The pIZT-mCherry vector transfected cells were used as a control. Three biologically independent samples were used.
The apoptosis morphology of BmN cells was determined by fluorescence microscopy. First, BV-EGFP at an MOI of 2 was added to the BmβGRP4 overexpressed BmN cells. Then, at 36 h p.i., all culture medium was removed, and the cells were fixed in 4% paraformaldehyde (PFA; Sangon) for 10 min at room temperature; after the removal of all 4% PFA, the cells were washed with PBS (pH 7.4) three times for 5 min each. After washing, the cell nuclei were stained with DAPI (Beyotime, Shanghai, China) in the dark for 10 min at room temperature. Finally, the cell morphology was examined using an inverted microscope DMi8 camera. The pIZT-mCherry vector transfected BmN cells were used as a control in this experiment. Three biologically independent samples were used.