A Fast And Versatile Method for Simultaneous HCR, Immunohistochemistry And Edu Labeling (SHInE)

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Advancements in sequencing technologies, especially single-cell transcriptomics, have increased the demand for in situ validation methods.
• Visualizing gene expression and protein localization is crucial for interpreting sequencing data and understanding cellular processes like proliferation.
• Complex tissues, often opaque or pigmented, pose significant challenges for traditional imaging and labeling techniques.

Purpose of the Study:

• To develop a versatile protocol for simultaneous in situ analysis of gene expression, protein localization, and cell proliferation.
• To demonstrate the compatibility of the developed protocol with tissue clearing techniques for deep tissue imaging.
• To provide a method for validating and contextualizing transcriptomic and proteomic data in complex biological samples.

Main Methods:

• Integration of in situ hybridization chain reaction (HCR) for transcript visualization.
• Incorporation of immunohistochemistry (IHC) for protein localization.
• Inclusion of 5-ethynyl-2'-deoxyuridine (EdU) labeling for cell proliferation assessment, combined with tissue clearing.

Main Results:

• The developed protocol successfully enabled parallel analysis of gene expression, protein localization, and cell proliferation.
• The method demonstrated compatibility with tissue clearing, allowing for imaging within complex and opaque biological structures.
• Proof-of-concept validation was achieved in bristleworm (Platynereis dumerilii) heads and trunks, showcasing its applicability.

Conclusions:

• This versatile protocol offers a powerful tool for multi-modal in situ analysis in challenging biological samples.
• It facilitates the integration of transcriptomic and proteomic data with cellular proliferation information.
• The method enhances the ability to visualize and interpret gene and protein expression within the complex tissue context.