Related Experiment Video
Updated: Aug 28, 2025
Free-floating Immunostaining of Mouse Brains
Published on: October 7, 2021
Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues
Aubrey M Kelly1, Brandon A Fricker1, Kelly J Wallace1
1Department of Psychology, Emory University, 36 Eagle Row, Atlanta, GA 30322, USA.
Abstract:
Identifying multiple proteins within the same tissue allows for assessing protein colocalization, is cost effective, and maximizes efficiency. Here, we describe a protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections. As opposed to slide-mounted immunohistochemistry, the free-floating approach results in less tissue loss and greater antibody penetration. Using distinct fluorophores for individual proteins, this protocol allows for visualization of three or more proteins within tissue sections. The protocol can be applied to other tissue types. For complete details on the use and execution of this protocol, please refer to Gonzalez Abreu et al. (2022).
Insights
This study presents a free-floating multiplex immunolabeling protocol for visualizing multiple proteins in rodent brain sections, enhancing efficiency and reducing tissue loss compared to traditional methods.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Assessing protein colocalization in tissues is crucial for understanding cellular mechanisms.
- Traditional slide-mounted immunohistochemistry can lead to significant tissue loss and limited antibody penetration.
- Multiplex protein identification offers cost-effectiveness and enhanced experimental efficiency.
Purpose of the Study:
- To describe a novel protocol for multiplex immunolabeling of proteins in free-floating rodent brain sections.
- To offer an improved method over slide-mounted immunohistochemistry for protein visualization.
- To enable the simultaneous detection of three or more proteins within tissue samples.
Main Methods:
- Development of a free-floating immunohistochemistry protocol for rodent brain sections.
- Utilizing distinct fluorophores for the simultaneous labeling of multiple proteins.
- Optimization for enhanced antibody penetration and reduced tissue loss.
Main Results:
- The free-floating approach minimizes tissue loss during the immunolabeling process.
- Greater antibody penetration was achieved compared to slide-mounted techniques.
- Successful visualization of three or more proteins within the same tissue section was demonstrated.
Conclusions:
- The described free-floating multiplex immunolabeling protocol is an efficient method for protein colocalization studies.
- This technique offers advantages in tissue preservation and antibody accessibility.
- The protocol is adaptable for application to various tissue types beyond rodent brain sections.
