Ti2 e a1 r

Maxillary or mandibular bones of mice were decalcified in 10% ethylene diamine tetraacetic acid (EDTA) (pH 7.4) at 4 °C for 21 d. For immunofluorescence staining, specimens were embedded in optimal cutting temperature compound (OCT), and sectioned into 10-μm thick sections. Sections were treated with 3% hydrogen peroxide and goat serum blocking, and then they were incubated with a primary antibody. The following primary antibodies were used: anti-Osterix (1:300; Abcam, Cambridge, UK), anti-RUNX2 (1:100; Abcam), anti-type I collagen (1:200; Boster Biological Technology, Wuhan, China), anti-Amelogenin (1:100; Santa Cruz Biotechnology, Dallas, TX), anti-CD31 (1:100; Affinity Biosciences, Changzhou, China). Sections were subsequently incubated by Alexa Fluor 488 IgG (1:1000; Invitrogen) and/or Alexa Fluor 568 IgG (1:1000; Invitrogen), and counterstained with DAPI (Sigma-Aldrich).
The images were captured using a confocal microscope (Nikon, TI2-E + A1 R, Japan), and processed using the ImageJ software (US National Institutes of Health, United States).

hUSLFs were seeded in a 24-well plate at the density of 2.4 × 10⁵ cells/ml and cultured in 5% CO₂ at 37 °C. Paraformaldehyde in PBS (Wuhan Servicebio Technology Co., Ltd., Wuhan, China) was used to fix the fibroblasts. To stain cytoskeleton F-actin, the cells were permeabilized with 0.1% TritonX-100 (Solarbio, Beijing, China) for 5 min, treated with phalloidin (5 μg/ml) (Wuhan Servicebio Technology Co., Ltd.) and incubated for 60 min at room temperature in the dark. DAPI (5 μg/ml, 100 μl/well) (Solarbio) was used to stain the nuclei for 10 min in the dark. The cell cytoskeleton was observed using a confocal laser scanning microscope (Ti2-E/A1R+; Nikon, Tokyo, Japan).

Based on the electron microscopic findings and to further investigate the mechanism of the bacteriostatic activity of AMPs on C. albicans, fluorescence-labeled peptides were employed to localize potential targets of the synthetic AMPs. Briefly, C. albicans cultures were treated with FITC-labeled AMPs at 4× the corresponding MIC value for 1 h and were then sequentially stained with propidium iodide (PI) and 4′, 6-diamidino-2-phenylindole (DAPI). Images were acquired with a confocal microscope (Ti2-E A1R+, Nikon, Tokyo, Japan).