FIGURE 7.

Optical clearing of HaCaT spheroids with Glycerol improves detection and quantification of cell nuclei. Upon growth to a diameter of approximately 300 μm, spheroids made of HaCaT keratinocytes were fixed, stained with anti-KI67 and DRAQ5, followed by optical tissue clearing or embedding as indicated and subsequent confocal whole mount microscopy. Semi-automated image segmentation was performed to detect and count KI67⁺ and DRAQ5⁺ nuclei. (A) Depicted are raw single optical sections and orthogonal views from central regions for both markers (KI67 – raw; DRAQ5 – raw) and corresponding segmented images (KI67 – segmented; DRAQ5 – segmented). In raw images, fluorescence signals of KI67 and DRAQ5 are shown in green and red, respectively. Different colors of segmented nuclei were used for better visual discrimination. Scale bars, 50 μm. B-C: Quantitative analysis of KI67⁺ (B) and Draq5⁺ nuclei (C) as a function of clearing/embedding protocol. Data show mean + SD; n ≥ 9; **p ≤ 0.01; ***p ≤ 0.001.