The aim of this study was to develop a procedure to remove the TO-PRO-3 fluorescent dye from tissue sections and restain with TO-PRO-3, still allowing calculation of DNA content and distribution by confocal laser scanning microscopy (CLSM). This would allow repeated measurements on the same tissue sections and prevents loss of tissue material from valuable clinical samples. Thick sections (14 lm) were cut from a paraffin block of adrenal tissue and stained using TO-PRO-3. Image stacks were acquired by CLSM. Thereafter, three destaining approaches were tested based on incubation, at different temperatures and durations, in the medium that is normally used to dissolve TO-PRO-3. The same areas were imaged again to measure residual fluorescence and were subsequently restained and imaged again. The intensity of the images acquired after initial staining and restaining were compared. A number of 3-D (texture) features computed after segmentation of nuclei were compared as well. The best destaining result was obtained by incubation of sections at 37°C in preheated medium twice for 20 min. On average, the 3-D feature values were comparable with those after initial staining. With the described protocol it is possible to remove TO-PRO-3 fluorescence from tissue sections that can successfully be restained with minimal influence on fluorescence intensity and nuclear chromatin distribution. ' 2007 International Society for Analytical Cytology Key terms confocal laser scanning microscopy; 3-D; image analysis; fluorescence staining; TO-PRO-3; restaining; tissue processing ASSESSMENT of nuclear DNA content and nuclear texture features by image analysis has proven to provide diagnostic and prognostic value in tumors from different sites (1,2). Confocal laser scanning microscopy (CLSM) presents the opportunity to perform 3-D DNA content measurements on intact cells in thick histological sections (3). This has major advantages over the established techniques of flow cytometry and conventional 2-D image cytometry on cytospins; dissociation of tissue with consequently loss of tissue architecture is not required, and inaccuracies caused by cutting or overlap as present in conventional image cytometry on thin tissue sections is almost completely avoided (4,5). In a previous study, we described the development of an optimal tissue processing technique for 3-D CLSM to establish DNA ploidy (6). As a measure of histogram quality, the coefficient of variation of the diploid peak was assessed. In addition, we have recently developed implementations to calculate nuclear chromatin texture features in 3-D (7). We showed the clinical relevance of measuring such 3-D nuclear chromatin texture to discriminate between benign and malignant cell nuclei from prostate cancer tissue (8). In all these studies, TO-PRO-3 was used for fluorescence staining as it provides optimal, specific, and quantitative staining of DNA, which is relatively stable (9,10). However, even TO-PRO-3 fluorescence does not remain constant in time because of bleaching and
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