A restaining method to restore faded fluorescence in tissue specimens for quantitative confocal microscopy.

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 microm) 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 degrees 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.