2.6. Quantification of UNC-45A Expression Levels via Immunofluorescence and via Immunohistochemistry

Quantification of immunohistochemistry was done on ImageJ using a slightly modified version of a previously described protocol [32]. Briefly, using whole slide scansions, regions of interest (ROIs) were drawn around the borders of the single elements analyzed before saving them into the ROI manager. Images were then converted into their RGB format and colors deconvolved using the color deconvolution function of ImageJ. Finally, the DAB signal was thresholded with a fixed upper limit of 70, and the mean grey values representing pixel intensity were obtained from individual ROIs. For quantification of immunofluorescence, pixel intensity was measured from the green channel after drawing the ROIs, and the background was subtracted without further image processing. At least three different areas were measured for each histological element and data compared using GraphPad Prism (version 8.4.3, San Diego, CA, USA).