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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

Whole mount immunohistochemistry uses antibodies to detect the spatial pattern of protein expression in the intact animal. The basic workflow of immunohistochemistry (depicted in Figure 1) involves breeding zebrafish, raising and preparing embryos, blocking non-specific antigens, using an antigen-specific primary antibody to target the protein of interest, detecting that primary antibody with a labeled secondary antibody, mounting the specimen, and documentin.......

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.......

Acknowledgements

Funding from NIH grant 8P20GM103436 14.

....

Materials

NameCompanyCatalog NumberComments
AgaroseFisher ScientificBP160-100
Aluminum foil, heavy dutyKirklandAny brand may be substituted
Anti-NMDA antibodyMillipore SigmaMAB363
Anti-phospho-Histone H3 (Ser10), clone RR002Millipore Sigma05-598
Anti-pan-AMPA receptor (GluR1-4)Millipore SigmaMABN832
Bovine serum albumin (BSA)Fisher ScientificBP1600-100
Calcium Nitrate [Ca(NO3)2]Sigma AldrichC4955
Centrifuge tubes, 1.5 mLAxygenMCT150C
Clear nail polishSally HansonAny nail polish or hardener may be subsituted
Depression (concavity) slideElectron Miscroscopy Sciences71878-01
DiaminobenzidineThermo Scientific1855920
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, E27.5 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 holderThermo Scientific59744015
Fluorescence compound microscopeLeica BiosystemsDMi8
Fluorescence stereomicroscopeLeica BiosystemsM165-FC
Glass coverslips 18 x 18Corning284518
Glass coverslips 22 x 60Thermo Scientific22-050-222
Glass slidesFisher Scientific12-544-4
GlycerolFisher ScientificBP229-1
Goat anti-mouse IgG Alexa 488InvitrogenA11001
HEPES solutionSigma AldrichH0887
Humid chamber with lidSimportM920-2
Hydrogen peroxide, 30%Fisher ScientificH325-500
Immunedge pap penVector labsH-4000
Insect pins, size 00Stoelting5213323
Magnesium Sulfate (MgSO4 · 7H2O)Sigma Aldrich63138
Mesh strainerOneidaAny brand may be substituted
MethanolSigma Aldrich34860
Methylene blueSigma AldrichM9140
Micro-tube cap lockResearch Products International145062
Microwave ovenToastmaster
Mouse IgGSigma AldrichI8765
Normal goat serumMillipore SigmaS02L1ML
Nutating mixerFisher Scientific88-861-044
ParaformaldehydeFisher Scientific04042-500
Pasteur pipettesFisher Scientific13-678-20C
PBTriton1% TritonX-100 in 1x PBS
Permount mounting mediumFisher ChemicalSP15-500
Petri dish (glass)Pyrex3160100
Petri dish (plastic)Fisher ScientificFB0875713
1-phenyl 2-thioureaAcros Organics207250250
Phosphate buffered saline (PBS), 10x, pH 7.4Gibco70011-044
Phosphate buffered saline (PBS), 1x1x made from 10x stock diluted in dH2O
Potassium Chloride (KCl)Sigma AldrichP9333
Potassium Hydroxide (KOH)FisherP250-500
Potassium Phosphate Monobasic (KH2PO4)Sigma AldrichP5655
PronaseSigma Aldrich10165921001
Proteinase KInvitrogenAM2544
Sodium Chloride (NaCl)Sigma AldrichS7653
Sodium Phosphate Dibasic (Na2HPO4)Sigma AldrichS7907
Spawning tank with lid and insertAquaneeringZHCT100
SuperBlock PBSThermo Scientific37515
Superfrost + slidesFisher Scientific12-550-15
Superglue gel3M Scotch
TNT100 mM Tris, pH 8.0; 150 mM NaCl; 0.1% Tween20; made in dH2O
Transfer pipetteFisher13-711-7M
Trichloracetic Acid (Cl3CCOOH)Sigma AldrichT6399
Tris BaseFisher ScientificS374-500
TritonX-100Sigma AldrichT9284
Tween20Fisher ScientificBP337-500
Ultrafine forcepsFisher Scientific16-100-121
Water, ultrapure/double distilledFisher ScientificW2-20

References

  1. Nasevicius, A., Ekker, S. C. Effective targeted gene 'knockdown' in zebrafish. Nature Genetics. 26 (2), 216-220 (2000).
  2. Gaj, T., Gersbach, C. A., Barbas, C. F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.

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