Inverted research microscope

Cells were serum starved in medium with 0.5% FCS for 24 h while sub-confluent, then harvested, resuspended in serum deficient medium, and seeded into 8 μm Transwell inserts at 1.25 × 10⁴ cells per insert and placed into 24-well companion plates containing DMEM + 10% FCS as a chemoattractant stimulus. After 4 h cells were fixed with 4% paraformaldehyde, stained with 3% crystal violet, and non-migratory cells removed from the inside of the insert with a cotton bud. Membranes were then imaged using an inverted research microscope (Olympus, Tokyo, Japan) with DP71 microscope digital camera (Olympus, Tokyo, Japan) with six images taken per membrane. Each set of images was taken in the same place for each membrane to minimize any bias and ensure consistency. Images were then analysed using ImageJ (Image Processing and Analysis in Java; US National Institutes of Health, Bethesda, MD, USA). Data are presented as the number of cells per field, which represents the mean of the counts across the six fields for each membrane. Replicate measurements (number of cells per field) were then combined and data analysed using unpaired t-tests.

Immunohistochemistry was performed in tumor tissues using the streptavidin-peroxidase (SP) method (Ultra Sensitive TM-SP) as described previously [32 (link)]. Scores were independently performed by two pathologists blinded to the identity of the tissue specimens as described [33 (link)]. The RUNX2 positive cells were photographed with Inverted Research Microscope (Olympus).