Technical development of senescence-associated β-galactosidase staining in frozen kidney tissue of Sprague Dawley rat.

To establish the optimal conditions for SA-β-galactosidase staining on frozen rat kidney tissue sections, we evaluated staining intensity, specificity, and consistency under several experimental conditions. We tested the effects of tissue freezing methods, fixative solutions and methods, section thickness, and duration of incubation time for SA-β-gal staining to obtain consistent results. This work was prompted by the emerging developments that strongly suggest the potential central roles of cellular senescence in aging and age-related diseases. To further examine the direct roles of senescent cell accumulation on age-related changes in various tissues and organs, it is essential to determine tissue localization and distribution, cell-type specificity, and direct correlations to histopathological changes. Furthermore, the recent advancements in molecular analyses, including spatial transcriptomics and MALDI-MSI spatial metabolomics, , on histology sections enable more in-depth and comprehensive analyses of underlying mechanisms to determine the roles that senescent cells play in age-related pathophysiology by integrating SA-β-gal stained tissue sections from the same samples. Based on our results, the following method showed the optimal SA-β-gal staining for frozen rat kidney sections: a) preparation of frozen blocks in OCT compound using a dry ice methanol bath; b) slides with both pre- and post-fixation method showed the most intense staining with clear tissue structural patterns; c) the staining areas and intensity were higher and specific with thicker (20 μm) tissue sections; and d) a 4-hour staining incubation period with both pre- and post-fixation method showed the optimal intensity and specificity. Although we established the optimal SA-β-galactosidase staining protocol with kidney, further evaluation is needed to determine the optimal staining conditions for other tissues. We believe that consistent and specific SA-β-galactosidase staining in frozen kidney sections will facilitate comparative analyses with new molecular imaging techniques to examine the exact roles of cellular senescence in aging and age-related pathology.