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

Josy Augustine; Madeleine R. Smith; Ryan Delaney; Precious O. Owuamalam; Guilherme Costa

doi:10.1016/j.xpro.2024.103510

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

Josy Augustine, Madeleine R. Smith, Ryan Delaney, Precious O. Owuamalam, Guilherme Costa^*

^*Corresponding author for this work

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Abstract

The developing vasculature of the post-natal mouse retina is a powerful model to discover mechanisms of vessel formation and to test modulators of neovascularization. We present a protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas enabling the detection of individual mRNAs in vascular endothelial cells. We describe procedures from initial retina preparation to smFISH and detection. Our approach offers simple steps to overcome challenges related to tissue permeabilization, mRNA and protein co-detection, and post-acquisition image processing.

Original language	English
Article number	103510
Number of pages	15
Journal	STAR Protocols
Volume	5
Issue number	4
Early online date	12 Dec 2024
DOIs	https://doi.org/10.1016/j.xpro.2024.103510
Publication status	Published - 20 Dec 2024

Keywords

single-molecule FISH
FISH
mouse retinal vasculature
vessel formation
retina - metabolism
developmental biology
single molecule imaging - methods
endothelial cells - cytology - metabolism
RNA, messenger - genetics - metabolism
animals
retinal vessels - metabolism - growth & development
mice
in situ hybridization, Fluorescence - methods
gene expression

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10.1016/j.xpro.2024.103510Licence: CC BY

Protocol for single-molecule FISH in the developing mouse retinal vasculature
Copyright 2024 the authors. This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Final published version, 5.57 MBLicence: CC BY

Cite this

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title = "Protocol for single-molecule FISH in the developing mouse retinal vasculature",

abstract = "The developing vasculature of the post-natal mouse retina is a powerful model to discover mechanisms of vessel formation and to test modulators of neovascularization. We present a protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas enabling the detection of individual mRNAs in vascular endothelial cells. We describe procedures from initial retina preparation to smFISH and detection. Our approach offers simple steps to overcome challenges related to tissue permeabilization, mRNA and protein co-detection, and post-acquisition image processing.",

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AU - Augustine, Josy

AU - Smith, Madeleine R.

AU - Delaney, Ryan

AU - Owuamalam, Precious O.

AU - Costa, Guilherme

PY - 2024/12/20

Y1 - 2024/12/20

N2 - The developing vasculature of the post-natal mouse retina is a powerful model to discover mechanisms of vessel formation and to test modulators of neovascularization. We present a protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas enabling the detection of individual mRNAs in vascular endothelial cells. We describe procedures from initial retina preparation to smFISH and detection. Our approach offers simple steps to overcome challenges related to tissue permeabilization, mRNA and protein co-detection, and post-acquisition image processing.

AB - The developing vasculature of the post-natal mouse retina is a powerful model to discover mechanisms of vessel formation and to test modulators of neovascularization. We present a protocol for single-molecule fluorescent in situ hybridization (smFISH) in whole-mount mouse retinas enabling the detection of individual mRNAs in vascular endothelial cells. We describe procedures from initial retina preparation to smFISH and detection. Our approach offers simple steps to overcome challenges related to tissue permeabilization, mRNA and protein co-detection, and post-acquisition image processing.

KW - single-molecule FISH

KW - FISH

KW - mouse retinal vasculature

KW - vessel formation

KW - retina - metabolism

KW - developmental biology

KW - single molecule imaging - methods

KW - endothelial cells - cytology - metabolism

KW - RNA, messenger - genetics - metabolism

KW - animals

KW - retinal vessels - metabolism - growth & development

KW - mice

KW - in situ hybridization, Fluorescence - methods

KW - gene expression

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DO - 10.1016/j.xpro.2024.103510

M3 - Article

SN - 2666-1667

VL - 5

JO - STAR Protocols

JF - STAR Protocols

IS - 4

M1 - 103510

ER -

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

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Keywords

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