Eclipse ti

Cells were transfected with an adenovirus expressing mCherry-GFP-LC3B fusion protein (AdPlus-mCherryGFP-LC3B) (C3012, Beyotime, Chinaper the manufacturer’s instructions. After adenovirus transfection at a multiplicity of infection (MOI) of 20 for 24 h, then we removed the adenovirus-containing culture medium and added the fresh complete culture medium to each well for another 24 h culture. The transfected cells were visualized using a confocal microscope (Eclipse Ti, NIKON, Tokyo, Japan). We calculated the LC3B punctate dots using ImageJ 1.50b. For actin microfilaments in Huh7 cells with or without AdPlus-mCherry-GFP-LC3B transfection, we washed cells in PBS twice, fixed them with 4% paraformaldehyde for 10 min at room temperature, permeabilized them with 0.5% Triton X-100 for 5 min, washed them three times in PBS, blocked them with 0.5% BSA for 30 min, and stained them with Texas Red®-X phalloidin (T7471, Invitrogen, USA) for 30 min and 4'6'-diamidino-2-phenylindole (DAPI, 760–4196, Roche, Germany) for 20 min at room temperature. Cells were sealed with 10% glycerol and then kept in the dark. We visualized the actin cytoskeleton (red) and nuclei (DAPI, blue) using a confocal microscope (Eclipse Ti, NIKON, Tokyo, Japan).

Rat BMSCs were seeded into 12-well TCPs at a density of 2×10⁵ cells/well, followed by coculture with a transwell system (Corning, New York, NY, USA) loaded with the five groups of scaffolds. On Day 7, each plate was rinsed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes. After being washed twice with distilled water, the cells were subjected to ALP staining (Beyotime, Shanghai, People’s Republic of China) according to the manufacture’s instruction. Stained cells were examined under a microscope (Eclipse Ti; Nikon). An ALP activity quantitative assay was also performed at the same time, exactly as in the described assay for the IC and BMP2 synergy. The relative ALP activity of each individual counterpart was assessed as an OD value at 405 nm per milligram of total protein and then normalized to the fold changes by using the control group SF/SBA15 as the reference value (set to 1) and expressed as the mean ± SD. Half of the culture medium was replaced at 3 days. In addition, on Days 14 and 28, fixed cells were stained with 1% Alizarin red (Sigma-Aldrich) for 10 minutes. The cells were then rinsed with water and viewed with an inverted light microscope (Eclipse Ti; Nikon). Half of the culture medium was replaced at 3, 7, 10, 14, 17, 21, and 24 days.

Imaging was carried out using either a Nikon EclipseTi microscope with a color camera for IHC or a Nikon Eclipse Ti confocal microscope for IF images. Images were acquired using a 10X objective lens to ensure coverage of tissue rather than in-depth visualization of a small region. Quantification was conducted automatically using Elements Analysis software provided with a Nikon microscope for both applications. Threshold was defined and maintained throughout all images for each application to ensure no bias was applied to the data. Thresholds were applied to exclude low and high outliers. The red/brown color from the HRP/DAB reactivity with the CCM2 antibody was quantified and averaged between the red and green channel quantification, and the fluorescent images were quantified for CCM1 and CCM3 using wavelength channels 488 and 647 nm, respectively.

All live cells were maintained at 37°C, 5% CO2 with a stage top incubator (Okolab) during imaging. For confocal microscopy, cells were imaged with a spinning disk confocal microscope (Eclipse Ti, Nikon) with a spinning disk (Yokogawa CSU-X, Andor), CMOS camera (Zyla, Andor), and either a 20x objective (Plano Fluor, 0.45NA, Nikonor a 60x objective (Apo TIRF, 1.49NA, oil, Nikon). For total internal reflection fluorescence (TIRF) microscopy, cells were imaged with TIRF microscope (Eclipse Ti, Nikon), 60x objective (Apo TIRF, 1.49NA, oil, Nikon) and EMCCD camera (iXON Ultra, Andor). Both microscopes were controlled with Micro-Manager. Images were analyzed and prepared using FIJI (Schindelin et al., 2012 (link)).