Open AccessQuantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy
Author(s) -
Isabell Smyrek,
Ernst H. K. Stelzer
Publication year - 2017
Publication title -
biomedical optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.8.000484
Subject(s) - immunostaining , light sheet fluorescence microscopy , staining , microscopy , spheroid , pathology , optical sectioning , stain , immunofluorescence , fluorescence microscope , histology , confocal microscopy , biomedical engineering , optics , fluorescence , biology , immunohistochemistry , microbiology and biotechnology , medicine , scanning confocal electron microscopy , physics , in vitro , biochemistry , antibody , immunology
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, 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.