3D Iterative Immunofluorescence Imaging on Whole-Mount Samples.
- 1Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Abstract
Multicellular organization relies on reciprocal interactions between molecular events, such as gene expression and protein state, and higher-scale properties, such as spatial patterning and tissue architecture. Understanding these processes requires methods that enable quantitative measurements at subcellular resolution, while maintaining the three-dimensional tissue organization. Conventional immunofluorescence imaging captures spatial information but is limited to the number of fluorescence markers that can be imaged simultaneously, whereas dissociation-based single-cell approaches can profile multimodal cellular states but lack positional information. Here, we describe 3D in toto iterative immunofluorescence imaging, termed 3D-4i, which enables up to ten-plex protein and protein state measurements in early zebrafish embryos. Leveraging sample immobilization on 96-well plates together with a gentle liquid handling system and high-content spinning disc confocal microscopy, this method comprises repeated rounds of antibody staining, optical clearing, confocal imaging, and antibody elution. Subsequent image analysis allows segmentation of nuclei and cells, extraction of quantitative single-cell features, and integration of molecular measurements with spatial context. Altogether, 3D-4i provides a scalable platform for investigating diverse biological processes in intact embryos, while maintaining both subcellular resolution and three-dimensional context. Key features • Enables volumetric, single-cell multiplexed protein imaging in intact early zebrafish embryos using iterative immunofluorescence imaging. • Compatible with automated liquid-handling and high-throughput spinning disc confocal microscopy. • Supports quantitative analysis of diverse biological processes at single-cell resolution.
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BibTeX
@article{wyss2026iterative,
title = {{3D Iterative Immunofluorescence Imaging on Whole-Mount Samples.}},
author = {Marvin F Wyss and Max Hess and Shayan Shamipour and Lucas Pelkmans},
journal = {Bio-protocol},
year = {2026},
month = oct,
doi = {10.21769/bioprotoc.5838},
url = {https://europepmc.org/article/MED/42859510},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - 3D Iterative Immunofluorescence Imaging on Whole-Mount Samples.
AU - Marvin F Wyss
AU - Max Hess
AU - Shayan Shamipour
AU - Lucas Pelkmans
PY - 2026
DA - 2026/10/05
JO - Bio-protocol
DO - 10.21769/bioprotoc.5838
UR - https://europepmc.org/article/MED/42859510
AB - Multicellular organization relies on reciprocal interactions between molecular events, such as gene expression and protein state, and higher-scale properties, such as spatial patterning and tissue architecture. Understanding these processes requires methods that enable quantitative measurements at subcellular resolution, while maintaining the three-dimensional tissue organization. Conventional immunofluorescence imaging captures spatial information but is limited to the number of fluorescence markers that can be imaged simultaneously, whereas dissociation-based single-cell approaches can profile multimodal cellular states but lack positional information. Here, we describe 3D in toto iterative immunofluorescence imaging, termed 3D-4i, which enables up to ten-plex protein and protein state measurements in early zebrafish embryos. Leveraging sample immobilization on 96-well plates together with a gentle liquid handling system and high-content spinning disc confocal microscopy, this method comprises repeated rounds of antibody staining, optical clearing, confocal imaging, and antibody elution. Subsequent image analysis allows segmentation of nuclei and cells, extraction of quantitative single-cell features, and integration of molecular measurements with spatial context. Altogether, 3D-4i provides a scalable platform for investigating diverse biological processes in intact embryos, while maintaining both subcellular resolution and three-dimensional context. Key features • Enables volumetric, single-cell multiplexed protein imaging in intact early zebrafish embryos using iterative immunofluorescence imaging. • Compatible with automated liquid-handling and high-throughput spinning disc confocal microscopy. • Supports quantitative analysis of diverse biological processes at single-cell resolution.
SN - 2331-8325
ER -