Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Summary

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Abstract

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red ^1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) ³. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy ^3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein ⁵. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Protocol

1. FIXATION Fix embryos overnight at 4°C with 4% paraformaldehyde (PFA) in PBS. Remove fix and wash 2 x PBS, 5 minutes each at room temperature (RT). Manually dechorionate embryos in PBS in a glass depression plate using watchmaker forceps. After dechorionation, embryos are transferred using a fire-polished glass Pasteur pipette as they may stick to polypropylene pipettes. Transfer embryos through a series of 25%, 50% and 75% methanol in PBS for 5 minutes each. Replace l…

Discussion

The protocol presented here works well with probes that give a clean strong signal after staining for 30-45 minutes in a typical alkaline phosphatase-mediated reaction. Prior to performing the fluorescent in situ hybridization protocol, we always test our probes using the standard non-fluorescent protocol (provided in supplemental material along with the probe synthesis protocol). We have had less success with the fluorescent in situ hybridization protocol when using weaker probes, but it may be possible in some cases….

Materials

Material Name	Type	Company	Catalogue Number	Comment
Paraformaldehyde 16% solution, EM Grade		Electron Microscopy Services	15710	Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at –20°C
Proteinase K, recombinant PCR grade		Roche	03115879001	Make a 20mg/ml stock solution in water. Store in 1ml aliquots at –20°C
Blocking Reagent		Roche	11096176001	Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at –20°C. Stable over multiple freeze/thaws.
Anti-Fluorescein-POD, Fab fragments		Roche	11426346910	Aliquot and store at -20°C. Keep one working aliquot at 4°C.
Anti-Digoxigenin-POD, Fab fragments		Roche	11207739910	As above.
TSA Plus Fluorescein system		Perkin Elmer	NEL741001KT	Prepare each vial of TSA reagent only when needed. Dissolve in 60μl DMSO. Store at 4°C.
TSA Plus Cyanine5 system		Perkin Elmer	NEL745001KT	As above
TSA Plus Cyanine3 system		Perkin Elmer	NEL744001KT	As above
TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)		Molecular Probes /Invitrogen	T20926	Dissolve tyramide reagent in 150μl DMSO, aliquot and store at –20°C.
RNase, DNase free		Roche	11119915001	Supplied as 500μg/ml solution
Propidium Iodide		Molecular Probes /Invitrogen	P-3566	Supplied as 1mg/ml solution in water.