Related Experiment Video
Updated: Oct 12, 2025
Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections
Published on: June 25, 2021
Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ
Ji Pang1,2, Nancy Thomas1, Dai Tsuchiya1
1Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA.
Insights
This study presents a novel histology protocol that enhances tissue integrity and optimizes molecular assays. The reproducible method may reduce animal use and lab costs.
Area of Science:
- Histology and Molecular Biology
- Biotechnology and Bioengineering
Background:
- Established histology protocols often struggle with maintaining tissue integrity.
- Optimizing tissue processing is crucial for accurate downstream molecular analyses.
Purpose of the Study:
- To introduce a robust and reproducible histology protocol.
- To enhance tissue integrity for improved histological staining and molecular assays.
Main Methods:
- Hartman's fixation, window technique, microwave-assisted processing, depigmentation, and antigen retrieval.
- ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence.
Main Results:
- The protocol effectively maintains tissue integrity.
- Optimized staining and multiplexing assays for molecular histology were achieved.
Conclusions:
- This novel protocol offers high reproducibility across laboratories.
- It has the potential to decrease animal usage and laboratory resource expenditure.
Abstract:
It can be challenging to maintain tissue integrity using established histology protocols. Here, we describe a protocol composed of Hartman's fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure. For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).
