A novel method for multiple labeling combining in situ hybridization with immunofluorescence

Area of Science:

• Neuroscience
• Molecular Biology
• Histology

Background:

• In situ hybridization (ISH) is crucial for studying mRNA expression in tissues.
• Standard ISH methods offer high specificity and preserve cellular morphology.
• Detecting specific cell populations in the central nervous system (CNS) requires sensitive techniques.

Purpose of the Study:

• To describe a novel method combining radioactive ISH and immunofluorescence on the same tissue section.
• To identify cell populations expressing specific mRNA transcripts in the CNS.
• To overcome limitations of existing double-labeling methods.

Main Methods:

• Radioactive in situ hybridization (ISH) combined with immunofluorescence.
• Utilized 4% paraformaldehyde, pH 9.5, for tissue fixation.
• Incorporated proteinase K digestion during the ISH procedure.

Main Results:

• The novel method completely protects hybridization signals from loss during immunoenzymatic reactions.
• Improved immunolabeling sensitivity was achieved due to proteinase K digestion.
• Multiple proteins and mRNA transcripts can be detected simultaneously in different cell populations.
• Tissue counterstaining is possible without compromising immunolabeling visualization.

Conclusions:

• This novel technique offers significant advantages over previous double-labeling methods.
• It is particularly useful for detecting low-abundance mRNAs like cytokines and chemokines in the CNS.
• The method facilitates the identification of specific cell populations in both intact and injured mammalian CNS.