Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy

Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full threedimensional information. In particular, the combination of optical clearing with light sheetbased fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verified in toto immunostaining protocols for large multicellular spheroids or for tissue sections have not been published. We present a method for the verification of immunostaining in three-dimensional spheroids. The analysis relies on three criteria to evaluate the immunostaining quality: quality of the antibody stain specificity, signal intensity achieved by the staining procedure and the correlation of the signal intensity with that of a homogeneously dispersed fluorescent dye. We optimized and investigated variations of five immunostaining protocols for three-dimensional cell biology. Our method is an important contribution to three-dimensional cell biology and the histology of tissues since it allows to evaluate the efficiency of immunostaining protocols for large three-dimensional specimens, and to study the distribution of protein expression and cell types within spheroids and spheroid-specific morphological structures without the need of physical sectioning. (C) 2017 Optical Society of America