We are trying to quantify macrophages signal in chicken intestine using ImageJ. We are applying an authomatic thereshold for green colour (macrophages) and trying to compare with total tissue area, but we are not sure if it is a correct and robust way. We took from 5 to 10 fields at 63x from each animal and need to know if there is a viable way of quantifying immune cells in tissue and comparing between different animals and groups. Intestines were frozen and cut with cryostat. Any advice?
Hi Lourdes, quantifying fluorescence is difficult in the best of situations.
Adding ImageJ to the mix creates more problems because applying a threshold is arbitrary and will generate false positive/negative regions depending how uniform the tissue sections are.
Plus, you will probably have autofluorescence in fixed tissues that is impossible to control for.
I have encountered this problem before and one way to get subjective values is to take pictures of random fields of fluorescent cells and use photoshop to count the pixels of positively stained cells.
Thanks a lot Steingrimur Stefansson, we are considering to do flux citometry instead of immunofluorescence...
I have no idea about what flux cytometry is all about, but good luck if you think that it will help.
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