Model ix70

The HL60/ABCG2 cells were cultured overnight in 24-well plates, then 0.4 µM tucatinib was added to each well for further incubation for 72 h. After washing with PBS, the treated cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.1% Triton X-100 for 10 min, both at room temperature. Subsequently, the cells were incubated with a monoclonal antibody against ABCG2 (cat. no. MAB4145; clone BXP-34; (1:500), overnight at 4°C. Following incubation, the cells were washed with ice-cold PBS and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (cat. no. A-10684; 1:1,000) for 1 h, at 4°C. Immunofluorescence images were obtained using an inverted confocal microscope, and 6–8 random microscopic fields (magnification, ×400; model IX70; Olympus Corporation) with IX-FLA fluorescence and a Charge Coupled Devices camera.

The skeletal muscle, small intestines, and brain tissues were treated with 4% paraformaldehyde at 4°C for 24 h. Then, the tissues were embedded in paraffin (Beyotime Biotech.) and cut into sections 4 μm thick. Tissue slices were subsequently de-paraffinized as previously described [23 ]. The tissue sections were used for HE staining assay as previously described [24 (link)]. Briefly, the above tissue sections were stained with hematoxylin for 10 min, followed by eosin staining for 30 s. Both hematoxylin and eosin staining were conducted at room temperature. The stained sections were imaged with an inverted fluorescence microscope (Model IX70; Olympus, Tokyo, Japan) and analyzed using the Medical Image Analysis System (HMIAS22000; Wuhan Champion Image Technology Co., Ltd., Wuhan, China).

Wound‐healing assays were carried out using the tip of a 1‐mL micropipette to make a straight scratch on a confluent monolayer of B16‐F10 cells in a 6‐well plate. Then, the cells were rinsed twice using PBS before adding the indicated concentrations of purified β₂‐GPI, recombinant D1, D4, D5, D1234, and D12345 polypeptides at 0 and 24 hours. Representative images of melanoma cell migration were photographed using an inverted phase microscope (Model IX70; Olympus, Tokyo, Japan). For all treatments, the wound at 0 hour was assigned as 100% and was compared with the percentage of wound healing at 24 hours. Photographs were taken using more than three fields per well and ImageJ software was used to calculate wound areas.

Before each experiment, the ends of a trabecula (400–1,000 µm long × 100−200-µm diameter) were trimmed and mounted between a force transducer (Model 403A; Aurora Scientific) and a high-speed length controller (Model 322C; Aurora Scientific). The experimental apparatus was placed on the stage of an inverted microscope (Model IX70; Olympus) fitted with a 40× objective and a CCTV camera (Model WV-BL600; Panasonic). A halogen lamp was used to illuminate the skinned trabecula. Bitmap images of the trabecula were acquired using an AGP 4X/2X graphics card and associated software (ATI Technologies) and were used to measure mean sarcomere length and fiber dimensions during activation and relaxation. All experiments were performed at 22°C and at a sarcomere length of ∼2.20 µm in pCa 9.0 solution. Changes in force and motor position were sampled (16-bit resolution, DAP5216a; Microstar Laboratories) at 2.0 kHz using SLControl software (http://www.slcontrol.com). All data were saved to computer files for subsequent analysis.