Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.
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.
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 screens using N-ethyl-N-nitrosourea (ENU) or other mutagens due to challenges in establishing reverse genetic techniques. Roughly two decades ago, morpholinos were first used in zebrafish to knockdown targeted genes1. Morpholinos are small antisense oligonucleotides that inhibit translation of target mRNA following microinjection into an embryo at an early developmental stage. A major weakness of morpholinos is that they are diluted as the cells divide and generally lose effectiveness by 72 hours post-fertilization (hpf). While morpholinos remain a powerful tool for zebrafish gene disruption, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPRs) are more recently being used to directly target the zebrafish genome2,3. These reverse genetic strategies, in combination with forward genetics and high throughput screens, have established the zebrafish as a powerful model to study gene expression and function.
The ability to study gene function generally requires an evaluation of the spatio-temporal distribution of gene or gene product expression. The two most commonly used techniques to visualize such expression patterns during early development are in situ hybridization (ISH) and whole mount immunohistochemistry (IHC). In situ hybridization was first developed in 1969 and relies on the use of labeled antisense RNA probes to detect mRNA expression in an organism4. In contrast, labeled antibodies are used in immunohistochemistry to visualize protein expression. The idea of labeling proteins for detection dates back to the 1930's5 and the first IHC experiment was published in 1941 when FITC-labeled antibodies were used to detect pathogenic bacteria in infected tissues6. ISH and IHC have evolved and improved significantly over the subsequent decades and are now both routinely used in the molecular and diagnostic research laboratory7,8,9,10,11. While both techniques have advantages and disadvantages, IHC offers several benefits over ISH. Practically, IHC is much less time consuming than ISH and is generally less expensive depending on the cost of the primary antibody. In addition, mRNA expression is not always a reliable metric of protein expression as it has been demonstrated in mice and humans that only about a third of protein abundance variation can be explained by mRNA abundance12. For this reason, IHC is an important supplement to confirm ISH data, when possible. Finally, IHC can provide subcellular and co-localization data that cannot be determined by ISH13,14,15. Here, we describe a step-by-step method to reliably detect proteins by immunohistochemistry in whole mount zebrafish embryos and larvae. The goal of this technique is to determine the spatial and temporal expression of a protein of interest in the whole embryo. This technology utilizes antigen-specific primary antibodies and fluorescently tagged secondary antibodies. The protocol is readily adaptable to use on slide-mounted tissue sections and for use with chromogenic substrates in lieu of fluorescence. Using this protocol, we demonstrate that developing zebrafish skeletal muscle expresses ionotropic glutamate receptors, in addition to acetylcholine receptors. NMDA-type glutamate receptor subunits are detectable on the longitudinal muscle at 23 hpf.
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…
The authors have nothing to disclose.
Funding from NIH grant 8P20GM103436 14.
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 |