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, 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.
Highlights
Multicellular tumor spheroids (MCTS) resemble characteristics of in vivo tumor tissue
It is essential to work with MCTS of an adequate size to study in vivo tumor characteristics
96-well microplates were coated with agarose to form a concave surface. 10,000 cells were seeded in 100 μl culture medium per well
Summary
Multicellular tumor spheroids (MCTS) resemble characteristics of in vivo tumor tissue. Both develop morphological specialties such as a proliferative, a quiescent and a necrotic zone [1,2], which differ in morphology and in their cellular response to drugs [3,4]. Morphological zones only develop when a spheroid reaches a minimum size. The spheroid diameter and the cultivation time influence the availability of oxygen and nutrients throughout the spheroid, which eventually lead to a reduction of cell survival in the core region [5,6,7]. The concentric layering in MCTS constitutes a diverse cellular microenvironment that shares similarities to some in vivo tumors. It is essential to work with MCTS of an adequate size to study in vivo tumor characteristics
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