Mayer s hematoxylin histological staining reagent

Migration and invasion assays were performed in 24-well cell culture plates with 8.0-µm-pore Transwell inserts (Corning, Inc., Corning, NY, USA). For the invasion assay, the membranes were coated with Matrigel (BD Biosciences, San Jose, CA, USA), and the subsequent steps were the same as those in the migration assay. HSC-2 cells (8 × 10³) and SCC-9 cells (4 × 10⁴) were transfected with siRNA-HIF-1α or siRNA-Par3 and cultured in inserts in Opti-MEM (Gibco) at 37 °C in the presence of 5% CO₂ overnight. The next day, Opti-MEM was added to the upper chamber and 10% FBS medium to the lower chamber, and the plates were incubated for 24 h. The inserts were fixed and stained with Mayer’s hematoxylin histological staining reagent (Dako, Santa Clara, CA, USA). After washing the inserts with PBS, migrated and invaded cells were counted under an inverted light microscope using an Olympus U-TV camera (Olympus, Tokyo, Japan). Migrated or invaded cells were counted in eight random fields. All assays were performed in triplicate.

To analyze the migration and invasion abilities of the OSCC cells, a 24-well plate cell culture insert system (8.0-µm pore size; Corning, Inc.) was used. To determine the invasion abilities of the OSCC cells, the inserts were precoated with 5% collagen mixed with 40 µl of reduced serum Opti-MEM (Gibco). After transfection with the negative control or miRNA-18a, the reduced serum Opti-MEM medium was added to the upper insert and culture medium containing 10% FBS was added to the lower plate for 24 h. The inserts were then fixed with methanol and stained using Mayer’s Hematoxylin Histological Staining Reagent (Dako, Santa Clara, CA, USA) for 20 min at room temperature. The noninvaded cells in the upper chamber were scraped out using a cotton swap, and the number of invaded cells was counted using an inverted light microscope (Olympus, Tokyo, Japan) to make observations. The images of the migrated and invaded cells were randomly selected from seven parts of each insert, and the number of cells was calculated using ImageJ. All experiments were performed at least in triplicate.