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I'm using I-solution software to quantify the strength of the immunohistochemical immunoperoxidase staining in tissue of stathmin protein. I have been trying two methods: One method is by using color threshold (usually having to use a slightly different threshold for each staining to reflect what is seen) and then binary conversion and then measuring the area of that.
The second method is check mean gray value of background. I'm leaning towards this method being better.
Any suggestions? Does anyone have any comments on good methods?
Thank you very much !
Using ImageJ or any other software for comparative morphometric analysis of histochemically stained sections, the first "must" is to analyze sections of the same thickness and treated for the whole steps of the histochemical procedure at the same time with the same baths. Second, if You use DAB and a light Hematoxylin for general background structures, it is fine in ImageJ split channels ("Image-Color-Split channels") and work on the Blue channel image, in order to increase labeled cells contrast lightening the background. Third, the equalization over 256 grey levels of the first working image has to be made manually, registering the limits of the equalization interval (e.g. 85-218) for normalization. The equalization interval has to be identically selected and applied for all samples to be compared. At this point the segmentation of the gray level images can be reliably performed with the tool "Image-Adjust-Threshold" (not Color threshold) until to the best selection of labeled cells. The chosen threshold value has to be applied, maintaining the same value for all the images. Last step before measurement of Area of interest evaluated over the whole full frame area (or over a defined area previously selected), use the tool "Process-Binary-Make binary. Best wishes!
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Dear professor Alberto Calligaro,
Thank you very much for your response!
I am using threshold value to define for neuron positive cell on Immunohistochemistry image. I have slightly, middle and strongly staining of labeled cells. Which threshold value of labeled cell is the best? Looking for your kind reply,
Many thanks in advance for your time and consideration.
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