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  3. Protocol for whole-tissue immunolabeling, optical clearing, and lightsheet imaging of c-Fos protein expression in unsectioned mouse brains

Protocol for whole-tissue immunolabeling, optical clearing, and lightsheet imaging of c-Fos protein expression in unsectioned mouse brains

  • STAR Protoc. 2025 Jun 20;6(2):103868. doi: 10.1016/j.xpro.2025.103868.
Koukou Fu 1,  Jing Yang 2
Affiliations

Affiliations

  • 1 State Key Laboratory of Membrane Biology, School of Life Sciences, Center for Life Sciences, IDG/McGovern Institute for Brain Research, Peking University Third Hospital Cancer Center, Peking University, Beijing 100871, China.
  • 2 State Key Laboratory of Membrane Biology, School of Life Sciences, Center for Life Sciences, IDG/McGovern Institute for Brain Research, Peking University Third Hospital Cancer Center, Peking University, Beijing 100871, China; Peking Union Medical College Hospital, Beijing 100730, China. Electronic address: jing.yang@pku.edu.cn.
PMID: 40455684 DOI: 10.1016/j.xpro.2025.103868
Abstract

The c-Fos protein has been broadly utilized as a marker of neuronal activity, and conventional immunohistochemistry to determine its expression relies on tissue sections. Here, we present a protocol to visualize the endogenous c-Fos protein in intact, unsectioned mouse brains responding to specific stimuli based on the immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) method. We describe steps for tissue harvesting, fixation, decolorization, and permeabilization followed by whole-tissue anti-c-Fos immunolabeling. We then detail procedures for tissue embedding and optical clearing for imaging by lightsheet microscopy. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Keywords

Biotechnology and bioengineering; Microscopy; Neuroscience.

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