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we will count the number of the co-stain cells or double staining cells using image J the first step is to color threshold the image in the threshold color window adjust the brightness saturation and the who sliders such that only the double staining cells are thresholded users can also choose to use different thresholding methods the threshold color can also be changed if needed the next step is to convert the image into binary if there are gaps inside the thresholded cells users can utilize the fill holes feature users can also utilize the water shed option to separate cell clumps into individual cells if needed to count the number of cells click on analyze and analyze particles in the analyze particles window set the particle size such that the undesirable cells can be excluded in the circularity and show option users can set the parameters of their choice next check on all the parameters required for cell counting once all the parameters are set click okay in the results wi