Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Summary

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Abstract

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Introduction

The zebrafish (Danio rerio) has emerged as a powerful model for the study of biological processes for several reasons including short generation time, rapid development, and amenability to genetic techniques. As a result, zebrafish are commonly used in high throughput small molecule screens for toxicological research and drug discovery. Zebrafish are also an attractive model for the study of developmental processes given that a single female can routinely produce 50-300 eggs at a time and the optically clear embryos develop externally allowing for efficient visualization of developmental processes. However, early research relied mostly on forward genetic scre....

Protocol

The procedures for working with zebrafish breeding adults and embryos described in this protocol were approved by the Institutional Animal Care and Use Committee at Murray State University.

1. Embryo Collection and Fixation

1. Prepare spawning tanks by placing adult zebrafish mixed sex pairs or groups in tanks with a mesh or slotted liner filled with system water overnight.
2. At lights on, change the spawning tank water for fresh system water to remove feces. Use a 14 h/10 h l.......

Discussion

Immunohistochemistry is a versatile tool that can be used to characterize the spatio-temporal expression of virtually any protein of interest in an organism. Immunohistochemistry is used on a wide variety of tissues and model organisms. This protocol has been optimized for use in zebrafish. Immunohistochemistry in different species may require different fixation and handling techniques, blocking solutions depending on species and the presence of endogenous peroxidases, and incubation times due to the thickness and compos.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Fisher Scientific	BP160-100
Aluminum foil, heavy duty	Kirkland		Any brand may be substituted
Anti-NMDA antibody	Millipore Sigma	MAB363
Anti-phospho-Histone H3 (Ser10), clone RR002	Millipore Sigma	05-598
Anti-pan-AMPA receptor (GluR1-4)	Millipore Sigma	MABN832
Bovine serum albumin (BSA)	Fisher Scientific	BP1600-100
Calcium Nitrate [Ca(NO3)2]	Sigma Aldrich	C4955
Centrifuge tubes, 1.5 mL	Axygen	MCT150C
Clear nail polish	Sally Hanson		Any nail polish or hardener may be subsituted
Depression (concavity) slide	Electron Miscroscopy Sciences	71878-01
Diaminobenzidine	Thermo Scientific	1855920
Embryo medium, Danieau, 30%			17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgS04, 0.18 mM Ca(NO3)2, 1.5 mM HEPES in ultrapure water.
Embryo medium, E2			7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO4, 75 uM KH2PO4, 25 uM Na2HPO4, 0.5 mM CaCl2, 0.35 mM NaHCO3, 0.5 mg/L methylene blue
Floating tube holder	Thermo Scientific	59744015
Fluorescence compound microscope	Leica Biosystems	DMi8
Fluorescence stereomicroscope	Leica Biosystems	M165-FC
Glass coverslips 18 x 18	Corning	284518
Glass coverslips 22 x 60	Thermo Scientific	22-050-222
Glass slides	Fisher Scientific	12-544-4
Glycerol	Fisher Scientific	BP229-1
Goat anti-mouse IgG Alexa 488	Invitrogen	A11001
HEPES solution	Sigma Aldrich	H0887
Humid chamber with lid	Simport	M920-2
Hydrogen peroxide, 30%	Fisher Scientific	H325-500
Immunedge pap pen	Vector labs	H-4000
Insect pins, size 00	Stoelting	5213323
Magnesium Sulfate (MgSO4 · 7H2O)	Sigma Aldrich	63138
Mesh strainer	Oneida		Any brand may be substituted
Methanol	Sigma Aldrich	34860
Methylene blue	Sigma Aldrich	M9140
Micro-tube cap lock	Research Products International	145062
Microwave oven	Toastmaster
Mouse IgG	Sigma Aldrich	I8765
Normal goat serum	Millipore Sigma	S02L1ML
Nutating mixer	Fisher Scientific	88-861-044
Paraformaldehyde	Fisher Scientific	04042-500
Pasteur pipettes	Fisher Scientific	13-678-20C
PBTriton			1% TritonX-100 in 1x PBS
Permount mounting medium	Fisher Chemical	SP15-500
Petri dish (glass)	Pyrex	3160100
Petri dish (plastic)	Fisher Scientific	FB0875713
1-phenyl 2-thiourea	Acros Organics	207250250
Phosphate buffered saline (PBS), 10x, pH 7.4	Gibco	70011-044
Phosphate buffered saline (PBS), 1x			1x made from 10x stock diluted in dH2O
Potassium Chloride (KCl)	Sigma Aldrich	P9333
Potassium Hydroxide (KOH)	Fisher	P250-500
Potassium Phosphate Monobasic (KH2PO4)	Sigma Aldrich	P5655
Pronase	Sigma Aldrich	10165921001
Proteinase K	Invitrogen	AM2544
Sodium Chloride (NaCl)	Sigma Aldrich	S7653
Sodium Phosphate Dibasic (Na2HPO4)	Sigma Aldrich	S7907
Spawning tank with lid and insert	Aquaneering	ZHCT100
SuperBlock PBS	Thermo Scientific	37515
Superfrost + slides	Fisher Scientific	12-550-15
Superglue gel	3M Scotch
TNT			100 mM Tris, pH 8.0; 150 mM NaCl; 0.1% Tween20; made in dH2O
Transfer pipette	Fisher	13-711-7M
Trichloracetic Acid (Cl3CCOOH)	Sigma Aldrich	T6399
Tris Base	Fisher Scientific	S374-500
TritonX-100	Sigma Aldrich	T9284
Tween20	Fisher Scientific	BP337-500
Ultrafine forceps	Fisher Scientific	16-100-121
Water, ultrapure/double distilled	Fisher Scientific	W2-20