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

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.