Quantitative analysis of focal adhesions

HSCs were stained with anti-vinculin antibody as described above at a dilution of 1:200. Olympus Fluoview Fv10i (Olympus) laser scanning confocal microscope was used to take Z-stack images of individual cells using a 60× objective lens. A protocol previously described (Horzum et al. 2014) was used to perform quantitative analysis of focal adhesions using Fiji. First, the images were converted to 16-bit images and a Z-projection with maximum intensity was performed. After that, the following commands were executed: “subtract background with a rolling ball radius of 50 pixels,” “enhance contrast by running CLACHE plug-in with block size = 19, histogram bins = 256, and maximum slope = 6,” “minimize background by applying mathematical exponential (exp),” and “adjust brightness and contrast automatically.” Then, we applied a Mexican hat filter (can be found here: http://big-www.epfl.ch/sage/soft/LoG3D) with a radius of 5. Next, we converted the image to a binary image using the threshold command. To calculate the focal adhesion aspect ratio (major axis/minor axis), we enabled “Fit Ellipse” in Analyze > Set Measurements, which allows the major and minor axis to be displayed. Finally, we executed the “analyze particles” command and collected quantitative statistics about focal adhesions (e.g., size, area, number, and aspect ratio).