I have seen many articles with bar graphs on different percentages of positive cells from immunofluorescent photos, but I need to know if to create these graphs it is only necessary to observe the image or there really is a specific intensity value to define which cell is positive or not. (I think this is the best method, but I can not find this methodology). Please, recommend me any document that you I consult. Thanks!!
Hi Daniela,
Setting a minimum intensity value to define positive cells is mandatory. I think you can set limits in ImageJ (Freeware). You should even be able to find some plug-ins that will help you count % positive cells.
With kind regards,
Ronald
Hi Daniela,
the intensity values you get depend on many factors (mainly laser intensity and image acquisition settings). This is why there is no general number for a minimal fluorescence intensity value that corresponds to positive cells.
A good protocol to count positive cells is (in my opinion) the following:
1. Make sure the samples you want to compare are stained together, to avoid differences in the quality of your immunofluorescence staining
2. Acquire all your images with the same laser, objective and imaging settings
3. Take one representative image and use ImageJ to set a threshold so that your positive cells are above the threshold and negatives are below. This is really very much like looking at your image and deciding what is the best value.
4. Analyse all your images with the same threshold settings.
In practice, this is not very different from looking at your images and counting cells; one does this to eliminate personal bias, that might bring you to include cells that are not so clearly positive in some of the samples.
Best of luck!
Hi Daniela:
I suggest you you to follow all recommendations given by Vanessa. I would like to add the use of a negative control (without the primary antibody), in order to set your aquisition configurations to zero (laser intensity, gaining, etc). These control will help you to discriminate between background and signal.
Wish you luck!!!!!!
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