ISH-IEM: a sensitive method to detect endogenous mRNAs at the ultrastructural level

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Simultaneous detection of mRNA and proteins at the ultrastructural level is crucial for understanding cellular processes.
• Existing methods may compromise cellular ultrastructure or lack simultaneous detection capabilities.

Purpose of the Study:

• To describe a protocol for the simultaneous detection of mRNAs and proteins at the ultrastructural level.
• To enable visualization of the localization of specific transcripts and their associated proteins within cells.

Main Methods:

• The protocol integrates in situ hybridization (ISH) with cryo-immunolabeling.
• It involves ultrathin frozen sectioning, hybridization with labeled RNA probes (DIG or biotin), antibody detection with protein A-gold (PAG), optional protein labeling, and transmission electron microscopy (immuno-electron microscopy - IEM).

Main Results:

• The technique allows for the simultaneous detection of endogenous, overexpressed, or injected RNAs and proteins.
• Cellular ultrastructure is preserved during the process.
• The protocol is also adaptable for mRNA detection on semi-thin frozen sections combined with immunofluorescence.

Conclusions:

• This combined ISH and cryo-immunolabeling method provides a powerful tool for ultrastructural localization of RNA and protein interactions.
• It has been successfully applied to reveal the localization of specific transcripts (e.g., gurken, oskar mRNA) and their mediating proteins in Drosophila oocytes.