In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

Summary

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

Abstract

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

Introduction

The zebrafish is a powerful vertebrate model for studies of development and genetics^1,2. Zebrafish and humans share high genetic homology (70% of the genes are shared with the human genome), which allows its use as a model for human diseases³. In zebrafish, it is quite common to detect the expression patterns of two genes and their spatial relationship. Immunohistochemistry was first used in 1941 to detect pathogens in infected tissues by applying FITC-labeled antibodies⁴. The target protein in the tissue section is first labeled with a primary antibody, and the ....

Protocol

All animal protocols were approved by the Institutional Animal Care and Use Committee of Nantong University (No. S20191210-402).

1. Collection of zebrafish embryos

1. Set up a pair of zebrafish in breeding tanks the night before the eggs are to be collected, one a transgenic zebrafish and the other an AB wild-type zebrafish (Tg (foxP2:egfp-caax) X AB wild-type or Tg (hb9:egfp) X AB wild-type) (see the Table of Materials). Use a diagonal plastic divider to separate the male and female to preclude physical access. The following day, fit the upper part of the breedi....

Results

This protocol can be used to simultaneously examine the expression pattern of one mRNA and one protein. Figure 1 shows the experimental workflow. The 5-HT2C receptor is a subtype of the 5-HT receptor bound by the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT). It is widely distributed in the central nervous system (CNS) and can significantly regulate a variety of brain functions, including appetite, mood, anxiety, and reproductive behavior¹³. The expression of.......

Discussion

This protocol proposes a combination of in situ hybridization and immunohistochemistry, an important step in the colocalization experiments on zebrafish embryos. This method serves as an easy and efficient way to simultaneously analyze one mRNA and one protein. In situ hybridization and antibody staining were performed on zebrafish embryos. In contrast to several protocols published previously^14,15,16, immunofl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488 secondary antibody	Invitrogen	A21202
Anti-Digoxigenin AP Fab fragments	Roche	11093274910
Anti-GFP antibody	Millipore	MAB3580
Blocking reagent	Roche	11096176001
Blocking solution-1	made in lab	N/A	Dissolve the blocking reagent in 1X MAB to a final concentration of 10% (wt/vol). Autoclave and store at -20 °C before use.
Blocking solution-2	made in lab	N/A	0.1% Triton X-100, 3% BSA, 10% goat serum in 1x PBS
BM purple	Roche	11442074001
Bovine Serum Albumin (BSA)	Sigma	B2064
CaCl2	Sigma	C5670
Citrate buffer	Leagene	IH0305
Citric acid	Sigma	C2404
Cryomold for tissue, 15 mm x 15 mm x 5 mm	Head Biotechnology	H4566
DEPC-Treated Water	Sangon Biotech	B501005
Digital camera, fluorescence microscope	Nikon	NI-SSR 931479
E3 embryo medium	made in lab	N/A	5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4
Formamide	Invitrogen	AM9342
Goat serum	Sigma	G9023
Heparin sodium salt	J&K Scientific	542858
HYB	made in lab	N/A	preHYB plus 50 µg/mL heparin sodium salt, 100 µg/mL ribonucleic acid diethylaminoethanol salt
Immunohistochemical wet box	Mkbio	MH10002
KCl	Sigma	P5405
Low profile leica blades	Leica	819
MABT (1x)	made in lab	N/A	0.1 M maleic acid, 0.15 M NaCl, 0.02% Tween-20, pH 7.5
Maleic acid	Sigma	M0375
Methanol	J&K Scientific	116481
Methylene blue	Macklin	M859248
MgSO4	Sigma	M2643
NaCl	Sigma	S5886
NTMT	made in lab	N/A	0.1M Tris-HCl, 0.1M NaCl, 1% Tween-20
OCT medium	Tissue-Tek	4583
PAP pen	Enzo Life Sciences	ADI-950-233
Paraformaldehyde, 4%	Abbexa	abx082483	made in lab in 1x PBS
PBST (1x)	made in lab	N/A	1x PBS plus 0.1% Tween-20
Phenylthiourea	Merck	103-85-5
Phosphate-buffered saline (10x)	Invitrogen	AM9624
preHYB	made in lab	N/A	50% formamide, 5x SSC, 9.2 mM citric acid (pH 6.0), 0.1% Tween-20
Proteinase K	Roche	1092766
Ribonucleic acid diethylaminoethanol salt	Sigma	R3629
RNase-free 1.5 mL tubes	Ambion	AM12400
SSC (20x)	Invitrogen	AM9770
SSCT (0.2x)	made in lab	N/A	0.2x SSC plus 0.1% Tween-20
SSCT (1x)	made in lab	N/A	1x SSC plus 0.1% Tween-20
Sucrose	Invitrogen	15503022
Triton X-100	Sigma	T9284
Tween-20	Sigma	P1379
Zebrafish	Laboratory Animal Center of Nantong University	N/A