Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining

Roger Atanga; Amalia S Parra; Julie G In

doi:10.1016/j.xpro.2022.101775

Efficient RNA and RNA-protein co-detection in 3D colonoids by whole-mount staining

STAR Protoc. 2022 Oct 25;3(4):101775. doi: 10.1016/j.xpro.2022.101775. eCollection 2022 Dec 16.

Authors

Roger Atanga¹, Amalia S Parra², Julie G In³

Affiliations

¹ Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of New Mexico, Albuquerque, NM 87131, USA. Electronic address: ratanga@salud.unm.edu.
² Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, NM 87131, USA.
³ Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of New Mexico, Albuquerque, NM 87131, USA. Electronic address: jgin@salud.unm.edu.

Abstract

Here, we describe a protocol to visualize RNA oligos and proteins independently or together using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence in human colonoids, expanding on previously published research. Whole-mount staining is used to preserve the colonoid structure and fix onto glass slides. We describe procedures for efficient plating, fixation, and preservation of the colonoids. This workflow can be adapted to 3D organoid models from other tissues or organisms. For complete details on the use and execution of this protocol, please refer to In et al. (2020).

Keywords: Antibody; Cell Differentiation; Cell culture; In Situ Hybridization; Microscopy; Molecular Biology; Organoids; Stem Cells.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Fluorescent Antibody Technique
Humans
In Situ Hybridization, Fluorescence / methods
Organoids*
RNA*

Substances

RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding