Quantification of IHC analysis

ImageJ and ImageJ plugin IHC profiler were applied for quantification of IHC staining analysis as reported.61 After importing images into the software, IHC profiler was used for color deconvolution, by which DAB brown stain was separated from Mayer’s hematoxylin counterstain. Then, images were changed to 8-bit grayscale type and inverted under the “Edit” menu of ImageJ. After inversion, the DAB-stained areas are bright, and unstained areas are dark. The mean intensity was measured using the “Measure” function of ImageJ. Three fields of each group were assessed.