Te2000s inverted microscope

Cells were plated in fibronectin-coated 6-well plates at a density of 1 × l0⁵ cells/well and incubated for 24 h. After treatment with ATRA at concentrations of 0.01, 0.1, 1, and 10 µmol/L for another 24 h, proteoglycan expression in rEHBMCs was determined by Alcian blue staining . Briefly, cells were washed three times with double-distilled water, fixed in 4% (w/v) paraformaldehyde (Sigma, St. Louis, MO, USA) for 30 min, and then stained with neutral red for 15 min. Cells were then rinsed three times with double-distilled water and stained with 1% (w/v) Alcian blue (Boster Co., Ltd., Wuhan, China) for 30 min. Thereafter, the cells were dehydrated using 95% ethanol. Samples were observed using a Nikon TE2000-S inverted microscope (Nikon, Japan), and images were imported into Image-Pro Plus software (version 6.0; Media Cybernetics, Bethesda, MD, USA) for quantification. The area of blue staining was counted as the quantitative result.

For determination of the distribution of p53 and p21, cells were fixed in ice-cold 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and permeabilized with 0.2% Triton Χ-100 (Shanghai Chemical Reagent Co., Ltd.) for 10 min at 40°C. Cells were treated with 3% H₂O₂ for 20 min and non-specific binding was blocked with 10% normal goat serum (Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 30 min at 37°C. Following washing with PBS three times, cells were incubated with mouse monoclonal anti-p53 (1:200; cat. no. AP062; Beyotime Institute of Biotechnology, Haimen, China) and rabbit polyclonal p21 antibodies (1:200; cat. no. sc-397; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C followed by 1 h incubation with goat anti-rabbit (1:64; cat. no. BA1105; Boster Biological Technology, Ltd.) and goat anti-mouse fluorescein isothiocyanate-conjugated (1:64; cat. no. BA1101; Boster Biological Technology, Ltd.) antibodies. Following counterstaining with DAPI (Invitrogen Life Technologies, Carlsbad, CA, USA), cells were visualized under the Nikon TE2000-S inverted microscope (Nikon, Tokyo, Japan).