Establishment of an optimal antibody concentration for HM embedded tissues. (a) Images of human tonsil sections embedded in an A4B4P4 HM (3 mm × 3 mm × 0.5 mm) before and after clearing, 10 days. (b) Confocal z-stack imaging of antibody titrations in tonsil tissues (25X), Ki67 (yellow, left), CD3 (red, right), and DAPI (blue). For each antibody titration, three different concentrations were used: Ki67 (1:20, 1:100, 1:1000), CD3 (1:10, 1:50, 1:500), DAPI (1:500), and displayed in 3D: XY axes view (left), XZ axes view (middle), and a respective 2D optical slice (right). The images of corresponding isotype controls stained at the optimal concentrations are showed in the last row. (c) The mean fluorescence intensity (MFI) of Ki67 or CD3 and their isotypes were quantified using Image J at different tissue depths. Each optical section has multiple selected areas (n = 5) used for MFI quantification and the images were plotted with mean and standard deviation values by GraphPad Prism 7.0.