Hi!
In our project we need to analyze a certain protein secretion by cell. We did a RT-qPCR but the time left is not enough for western blot. We had the immuno-stained fluorescent images of the samples and gonna use the images as a quantification of the secreted protein using ImageJ. I checked several algorithm online but was still not sure how to canonically present the quantification as a measurement of the target protein. Could somebody offer more information and give some papers that use quantification of fluorescence in image as measurement of practicle amount?
Thanks!
Hello, certainly the best way to do semi-quantitative or quantitative immunofluorescence is by co-labeling with a control protein which should be the same for all. It is easier when quantifying intracellular proteins because you can correlate them with just DAPI labeling. Since you want to quantify a secreted protein I assume it means it is not inside the cell, although you may still be able to relate it with a control protein, maybe the methods in this paper will be helpful Article Voluntary running rescues adult hippocampal neurogenesis aft...
Hi!
In my personal experience, I used the program Fiji to quantify the immunostainig of a particular protein. This program enables you to calculate the tissue area, the positive area and so on, thus permitting you to normalize the positive area on tissue area (if you have tissue sections like heart setions, you might subtract the not-tissue area from the total area of your picture, thus obtaining the real tissue area). This method, however, is very long, but you can use it especially if the number of the pictures that you want no analyze is not too high. I learnt this method at Professor Sironi's laboratory (University of Milan), so maybe you can find more information searching online Laura Castiglioni/Luigi Sironi papers.
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