Ti s inverted microscope

On the third day of embryo development (D3), laser-assisted hatching (AH) was
performed on the zona pellucida to facilitate the hatching of the cells to be
biopsied. Only blastocysts categorized as grade 3 or better were biopsied.
During biopsy, six to ten trophectoderm cells were harvested.
All biopsies were performed using a Nikon Ti-S inverted microscope. An OCTAX
laser was used in the procedures. The blastocysts were biopsied on plates
containing three 10µL drops of Gmopsplus (Vitrolife, Sweden) covered with
mineral oil (Irvine Scientific).

After deparaffination and microwave antigen reparation, sections were pretreated with 3% H₂O₂ for 20 minutes to reduce the endogenous peroxidase activity. Then the sections were ordinally incubated with the blocking buffer (10% normal goat serum) at room temperature for 1 hour, then with primary antibodies of 4-hydroxynonenal (HNE) (1/100 dilution; Abcam, Cambridge, UK) at 4°C overnight, followed with biotinylated secondary antibody (1 : 200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and avidine-biotinylated peroxidase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) at room temperature for 1 hour. Coloration of sections was processed with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) and finished with distilled water. After counterstaining with hematoxylin for 30 seconds, sections were dehydrated with graded ethanol, cleared with dimethylbenzene, and mounted with neutral gums. Figures were captured with a Ti-S inverted microscope (Nikon, Japan) and analyzed with Image-Pro Plus software (version 6.0, Media Cybernetics, USA).

Calcium imaging using fura-2 was adapted from a previously published protocol with minor modifications46 (link). Briefly, ASMCs were loaded with 2 μM fura-2-AM (Thermo Fisher) and 24 h old serosal media from HBECs or media containing recombinant BPIFA1 at 37 °C for 1 h. ASMCs were then washed with a standard Ringer's solution (101 mM NaCl, 12 mM NaHCO₃, 1.2 mM MgCl₂, 1.2 mM CaCl₂, 0.2 mM KCl, 24 mM HEPES, 10 mM glucose, pH 7.4) or with Ca²⁺-free Ringer's solution as indicated. Cultures were then placed in the Ringer's solution, and images were collected with a 40 × 1.4 NA Plan Fluor oil objective on a Nikon Ti-S inverted microscope. Fura-2 fluorescence was acquired alternately at 340 and 380 nm (emission >450 nm) using LUDL filter wheels, obtained with an Orca FLASH 4.0 CMOS camera (Hammamatsu) and controlled with HCImageLive software (Hammamatsu). Cell bodies were identified as individual regions of interest (ROIs). Background subtraction was performed using a cell-free region as the background region. Signals were converted to relative changes (F/F₀) where F₀ was the ratio of the average fluorescent intensity (340/380) of ROIs at 0 time point. Typically, 20 cells per coverslip were recorded. ΔF/F₀ represents average peak fluorescent intensity changes of three independent experiments.

To test the effect of CQD-1 on hBMSCs’ differentiation, hBMSCs were cultured in osteogenic medium (OM) containing 10 mM β-glycerophosphate, 100 μM l-ascorbic acid 2-phosphate, and 10 nM dexamethasone (Sigma). Cells were cultured in the presence of PBS (Control group), OM (OM group), 1 μg mL⁻¹ CQD (OM + 1 μg mL⁻¹ CQD group), 10 μg mL⁻¹ CQD (OM + 10 μg mL⁻¹ CQD group), and 100 μg mL⁻¹ CQD (OM + 100 μg mL⁻¹ CQD group) for 7 d and 14 d. To observe mineralized nodules, hBMSCs were stained with 1% Alizarin Red S (Sigma) for 15 min at RT to detect mineral nodules after being fixed with 4% PFA for 1 h at 4 °C, after 7 d and 14 d incubation with conditioned medium. The stained cells were observed under the TI-S inverted microscope (Nikon, Tokyo, Japan) at 10× magnification. Experiments were performed in triplicate and repeated at least 3 times.