please I need your help in using ImageJ software for the quantification and assesment of brain blood capillaries in a different brain sections for immunohistochemistry for the evaluation of different treatments effects on the brain blood vessels and capillaries!
What kind of assessment are you doing? Blood vessel size or diameter? I have used image j for this before using a grid plugin and the measurement tools (and of course some sort of method of random sampling for unbiased results). Also, you will need to have z-stacks of your sections in order to accurately measure the size of BV's as they aren't 2D...
Hi Monica yes I'm trying to measure the size and quantify the numbers in each image.
please if you can provide me with the protocol you used for that
thanks
sweilem
Hey Sweilem,
Sorry for the late reply! You can download a grid plugging for fiji image J just by doing a google search. I can't remember the specific one I got... When you download it, you can open your images and then place the grid over your image, you can choose for it to be randomly placed or placed the same for each image.
I took 10 randomly selected z stacks for each section I was measuring. I also did 10 images per stack, the same width apart. You open each of the ten images per stack and analyse each image.
Depending on the number of vessels and density per image, you can select how many of the grids you will measure,, and you can also specify the grid size (you may want them larger or smaller etc). I chose to sample every 5th square for example, and measured only what was within those squares to limit bias.
To measure blood vessel diameter, I would draw a straight line along the vessel first, and then measure perpendicular to this line. You can then choose "Analyse-->measure" and it will save the widths. You can choose to only measure one vessel per square, or five per square, or only what lies in the centre of the field- its up to you.
Then once you get all your numbers you can pop them into excel and then prism or whatever statistical package you use to get stats and compare between sections. This is what I did anyway!
Hope that helps you!
Thank you so much Monica I will follow the protocol you suggested and I will let you know.
Thanks again for this very helpful response,
bests
Sweilem
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