<em>In Situ</em> Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

Jiaqi Wang; Rui Chai; Xiaoxia Fang; Jiajia Gu; Wanqing Xu; Qi Chen; Gang Chen; Shunxing Zhu; Yan Jin

doi:10.3791/63715

In Situ Hybrydyzacja połączona z immunohistochemią w kriosekcji zarodków danio pręgowanego

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

In Situ Hybrydyzacja połączona z immunohistochemią w kriosekcji zarodków danio pręgowanego

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07:36 min

March 3, 2022

DOI: 10.3791/63715-v

Jiaqi Wang*^1,2, Rui Chai*³, Xiaoxia Fang², Jiajia Gu⁴, Wanqing Xu², Qi Chen¹, Gang Chen^2,3, Shunxing Zhu⁵, Yan Jin¹

¹School of Life Sciences,Nantong University, ²Medical School,Nantong University, ³Key Laboratory of Neuroregeneration of Jiangsu Province and Ministry of Education, Co-Innovation Center of Neuroregeneration,Nantong University, ⁴Nantong Maternal and Child Health Care Hospital, ⁵Laboratory Animals Center,Nantong University

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Overview

This protocol outlines a method for obtaining images by integrating in situ hybridization and immunohistochemistry in zebrafish embryonic sections. It enables the simultaneous detection of mRNA and protein expression patterns, particularly useful in situations with limited antibody availability.

Key Study Components

Area of Science

Neuroscience
Developmental Biology
Gene Expression Analysis

Background

In situ hybridization allows for the localization of specific mRNAs.
Immunohistochemistry enables visualization of protein expression.
This dual approach is particularly beneficial for analyzing multiple genes simultaneously in zebrafish.
Zebrafish are a popular model organism for developmental studies.

Purpose of Study

To develop a protocol for visualizing both mRNA and protein in zebrafish embryos.
To address challenges related to antibody availability.
To facilitate the study of gene expression patterns during embryonic development.

Methods Used

The study employs a combination of in situ hybridization and immunohistochemistry.
The biological model consists of zebrafish embryos at various developmental stages.
Embryos are fixed, dehydrated, and subjected to hybridization with specific probes.
Immunostaining with primary and secondary antibodies follows the hybridization process.
Cryosectioning is implemented to prepare the embryos for imaging analysis.

Main Results

Gene expression of the 5-HT2C receptor and insm1a was successfully detected in specific zebrafish neuron populations.
The method demonstrated the simultaneous visualization of mRNA and protein in transgenic zebrafish.
Key insights into gene expression dynamics in the central nervous system were revealed.

Conclusions

This protocol effectively combines in situ hybridization and immunohistochemistry to enhance gene expression analysis.
The approach supports developmental neuroscience research by enabling comprehensive insights into gene interactions.
It provides a valuable tool for understanding neuronal mechanisms and gene regulatory networks in zebrafish models.

Frequently Asked Questions

What are the advantages of using zebrafish for this study?

Zebrafish embryos are transparent, allowing for easy observation of developmental processes and gene expression in real-time.

How is the hybridization process implemented?

Embryos are pre-hybridized and incubated with a heat-activated probe to facilitate specific binding to target mRNA.

What types of data are obtained through this method?

This protocol provides data on mRNA and protein localization, revealing expression patterns critical for developmental biology studies.

Can this approach be adapted for other model organisms?

While this protocol is optimized for zebrafish, the methodology might be adapted with considerations for the specific requirements of other organisms.

What are the key limitations of this method?

Challenges may include the specificity of probes and antibodies used and the intricacies of hybridization conditions that may vary for different targets.

How can the findings impact the understanding of gene regulation?

By visualizing gene expression concurrently, the method provides insights into how genes may coordinate with each other during development and cellular functions.

Ten protokół opisuje, jak uzyskać obrazy poprzez połączenie hybrydyzacji in situ i immunohistochemii odcinków embrionalnych danio pręgowanego. Przed kriosekcją przeprowadzono hybrydyzację in situ, a następnie barwienie przeciwciałami. Przydatne jest wykrycie wzorców ekspresji dwóch genów u danio pręgowanego, jeśli występuje niedobór przeciwciał.

Protokół ten wykorzystuje połączenie hybrydyzacji in situ i immunohistochemii i służy jako łatwy i skuteczny sposób jednoczesnej analizy jednego mRNA i jednego białka. Protokół ten ma zastosowanie do zarodków w różnych stadiach, skrawków tkanek o różnej grubości i różnych narządów. Na początek utrwal około 10 zarodków za pomocą 0,5 do 1 mililitra świeżego 4% paraformaldehydu w 1,5-mililitrowej probówce do mikrofugi w temperaturze czterech stopni Celsjusza przez noc przez 12 do 14 godzin.

Stopniowo odwadniaj zarodki, przemłukując je 25, 50 i 75% metanolem w PBST kolejno przez pięć minut w temperaturze pokojowej. Następnie myj zarodki w 100% metanolu przez pięć minut w temperaturze pokojowej. Inkubuj zarodki w 100% metanolu w temperaturze minus 20 stopni Celsjusza przez co najmniej noc, przez 12 do 14 godzin.

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