title: Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy.
abstract: Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verifiedin totoimmunostaining 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.
authors.affiliation: Buchmann Institute for Molecular Life Sciences, Goethe Universität, Frankfurt am Main, 60438, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Smyrek
authors.affiliation: Buchmann Institute for Molecular Life Sciences, Goethe Universität, Frankfurt am Main, 60438, Germany.
authors.forename: E H K
authors.initials: EH
authors.lastname: Stelzer
keywords: (170.6900) Three-dimensional microscopy; (180.2520) Fluorescence microscopy