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I am working with lung slices treated with test compounds A and B and later stained with DHE and PECAM (CD31), to understand if the test compounds A and B increase the ROS relative to each other and if so, is ithe increase/decrease in DHE staining intensity specific to PECAM lined endotheial cells?
To do this, I use a Carl Zeiss LSM 510 microscope and get the raw images of all planes. I use Image J to construct the stacks with DHE (red) and PECAM (green) pseudocoloured. My questions are:
1. What is the best way to get integrated intensity of DHE staining alone, across the entire image from all the stacks, using Image J?
2. Is there a method to selectively measure DHE fluorescene from PECAM-lined endothelial cells and not from other cells?
3. When I go to Image, Colour, RGB split: it will give me red, green and blue windows. Can I take the red window integrated intensity as a measure of DHE staining?
Please take some time to give your suggestions.
Thanks in advance
Bodiga
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