2 4 amidinophenyl 6 indolecarbamidine dihydrochloride

hADSCs (at a density of $5\times {10}^4$ cells/well) were seeded on 24-well culture plates (n = 4) and incubated in OM containing 20 µM GNPs, SPVD, or VGNPs for 10 days. The cells were washed with DPBS and fixed in 3.7% formaldehyde at room temperature for 30 min. They were then washed with DPBS twice again. After washing, the cells were permeabilized with 0.1% Tween #20 (Yakuri Pure Chemicals Co. Ltd., Kyoto, Japan) for 10 min and blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, USA) in PBS (GIBCO, Grand Island, USA) for 1 h at 4°C. A monoclonal anti-osteocalcin antibody (sc-74495, Santa Cruz, USA) was used at a dilution of 1:100. Goat anti-mouse IgG-PE (sc-3738, Santa Cruz, USA) secondary antibody was used at a dilution of 1:500. Nuclei were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Sigma-Aldrich, St. Louis, USA). The cells were examined by microscope digital camera.

Formalin-fixed, paraffin-embedded (FFPE) tissue processing and immunofluorescence testing were performed according to the methods described in a previous report [16 (link)]. Briefly, FFPE slides were deparaffinized and rehydrated before being placed in citric buffer (pH 6) and heated in a microwave at 800W for 30 min. Thereafter, they were washed 3 times (5 min each) with distilled water. The slides were then blocked with 1% BSA in Tris-buffered saline with Tween (TBST) for 30 min at room temperature (RT). Rabbit serum was diluted in 1% BSA in TBST to 1:200, dropped onto a slide, and incubated for 2 h at 37°C. In addition, normal goat serum diluted with TBS (1:5) was dropped onto the slide for 30 min at RT. After washing 3 times (5 min each) with TBST, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Thermo Scientific, USA), diluted to 1:200, was dropped onto the slide and incubated for 1 h at RT. The nuclei were then counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Sigma Aldrich, USA) for 10 min at RT and the slides were observed under a fluorescence microscope (Zeiss, Germany).