I have performed an immunostainning in rat brain brain cortex in order to see new blood vessels formation. In order to cuantify it, I have densitometered the images following these steps in ImageJ

1. Image>type>8-bit

2. Select area of interest

3. Add selection: image>overlay>add selection

4. Image>overlay>To ROI manager

5. Measure; area and grey intensity.

Then the grey intensity/area= IOD

Am I following the correct steps? Is there any other method to measure angiogenesis in rat brain cortex?

Thank you!

Sara