RNA Detection

Chapter 11

Detection of mRNA and Associated Molecules by ISH-IEM

on Frozen Sections

Catherine Rabouille

Abstract

The use of tagged RNA probes to directly hybridize frozen sections of chemically fixed tissues, followed by
the tag detection with specific antibodies and gold conjugates form the core of the in situ hybridization
(ISH)-immunoelectron microscopy (IEM) method that we have developed and successfully used to detect
endogenousgurkenandbicoidmRNAs inDrosophilaoocytes.

Key wordsIn situ hybridization, Electron microscopy, Frozen sections, Immunoelectron microscopy,

ISH-IEM, Ultrastructure, mRNA detection

1 Introduction

Cell polarization is established by the differential localization of

proteins. This can occur through their posttranslational targeting

mediated by protein domains interacting with specific effectors. It

can also occur through localized translation followed by the asym-

metric cellular localization of their mRNAs [1]. In this regard, the

best-studied examples of localized mRNAs aregurken,bicoid, and

oskarin the developingDrosophilaoocyte [2, 3].

Determining protein localization at the ultrastructural level at

the electron microscopy (EM) level is classically achieved by

immuno-EM on ~70 nm ultrathin frozen sections of chemically

fixed tissues that are obtained by a process called cryo-sectioning.

These preserve both the cell ultrastructure and protein antigenicity.

Primary antibodies against specific antigens are then used that are

detected by gold conjugates [4].

On the other hand, mRNA localization is achieved by RNA in

situ hybridization (ISH) that is a method of choice for mRNA

detection in cells or tissues. This uses tagged antisense RNA strands

(RNA probes) that specifically hybridize to their complementary

mRNAs. The tag (digoxigenin (DIG) or biotin) is then visualized

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_11,©Springer Science+Business Media LLC 2018

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