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Protocol for single-molecule FISH in the developing mouse retinal vasculature.

Josy Augustine1, Madeleine R Smith1, Ryan Delaney2

  • 1Wellcome-Wolfson Institute for Experimental Medicine, Queen's University, Belfast BT9 7BL, UK.

STAR Protocols
|December 13, 2024
PubMed
Summary

This study introduces a single-molecule fluorescent in situ hybridization (smFISH) protocol for whole-mount mouse retinas. This method allows detailed analysis of mRNA in vascular endothelial cells, aiding neovascularization research.

Keywords:
developmental biologygene expressionin situ hybridization

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Area of Science:

  • Ophthalmology
  • Developmental Biology
  • Molecular Biology

Background:

  • The post-natal mouse retina is a key model for studying blood vessel formation (vasculature development).
  • Understanding neovascularization mechanisms is crucial for treating various eye diseases.

Purpose of the Study:

  • To present a detailed protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas.
  • To enable the detection of individual messenger RNAs (mRNAs) within vascular endothelial cells.

Main Methods:

  • A protocol for smFISH in whole-mount mouse retinas is described.
  • Procedures cover retina preparation, smFISH, and signal detection.
  • The method addresses challenges in tissue permeabilization and mRNA/protein co-detection.

Main Results:

  • The smFISH protocol allows for the visualization and quantification of individual mRNAs in retinal vascular endothelial cells.
  • The approach simplifies complex steps in molecular analysis of retinal vasculature.
  • It facilitates overcoming common technical hurdles in imaging and molecular detection.

Conclusions:

  • This smFISH protocol provides a robust method for studying gene expression at the single-molecule level in retinal vasculature.
  • It serves as a valuable tool for investigating vessel formation and neovascularization.
  • The protocol enhances the utility of the mouse retina model for vascular research.