Choroid Sprouting Assay to Study Ocular Microvascular Proliferation Ex Vivo

Add 30 microliters of the thawed BME into the center of each well of a 24-well tissue culture plate. Make sure that the droplet of BME forms a convex dome at the bottom of the plate without touching the edges.

Place the tissue in the middle of the BME. Do not flatten the choroid explant. Let the tissue expand within the BME. Incubate the plate at 37 degrees celsius for 10 minutes to let the gel solidify. Then, add 500 microliters of complete classic medium into each well. Change the classic medium every other day.

Choroid sprouting can be observed after three days with a microscope. Open the choroid sprouting image with Image J and check "Image | Type | and 8-bit" with grayscale. Then optimize the contrast by selecting "Image | Adjust | Brightness/Contrast" and adjusting it.

Use the "Magic Wand" function to outline and remove the choroid tissue, which are present in the center of the sprouts. Remove the background of the image with the free selection tools.

Next, go to "Image | Adjust | Threshold " and use the "Threshold" function to define the microvascular sprouts against the background and periphery. Click "F2" and a summary will appear. Click "Save" to save an image of the selected area in the same folder as the original image for future reference.

After a group of samples is measured, copy the recorded data for analysis.