Tetramethylrhodamine phalloidin

Sections of the scaffolds (300 µm-thick) containing the HGFs were deparaffinized with xylene. Following rehydration with descending ethanol series, the samples were incubated for antigen retrieval in a microwave oven with EDTA buffer at pH 8.0 for 30 min at 95°C, followed by fixation with 4% paraformaldehyde for 30 min at room temperature. Immunofluorescence analysis was performed to detect fibrillar actin (F-actin) and nuclei. Structures of F-actin were detected using tetramethylrhodamine-phalloidin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) staining solution at a concentration of 100 nM and incubated for 30 min at room temperature. Nuclei were stained with DAPI at 10 µg/ml for 30 sec at room temperature (Sigma-Aldrich; Merck KGaA), according to the manufacturer's protocol. After washing with PBS, samples were examined by confocal microscopy (fv-500; Olympus Corporation).

The cell viability of irradiated osteocytes was detected using a Cell Counting Kit-8 assay (CCK8; Dojindo Molecular Technologies, Inc.). Briefly, osteocytes seeded in 96-well plates (3×10³ cells/well) were subsequently treated with irradiation (0, 2, 4 or 8 Gy), followed by incubation for 1, 3 or 5 days. CCK8 reagent was added at 10%, and incubated for 2 h at 37°C. The absorbance at 450 nm was examined using a microplate reader (Bioteck), and was considered to indicate cell viability.
The morphological changes including dendrite-like synapse and cytoskeleton in the irradiated osteocytes were examined by staining with tetramethyl rhodamine-phalloidin (Beijing Solarbio Science & Technology Co., Ltd.) for F-actin and with DAPI (Dojindo Molecular Technologies, Inc.) for nuclei. Each staining step was processed at room temperature and incubated for 2 h in the dark. The number and dendritic length of osteocytes were quantitatively measured using SimplePCI software (HCImage, SimplePCI 6.6). Six random fields were chosen in three biological replicates, and representative images were captured using a Leica fluorescent microscope (Leica Microsystems, GmbH) with a magnification of ×100. The length of the dendrites was calculated by total length of dendrites/number of dendrites per cell.