Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Summary

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Abstract

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Protocol

1. Riboprobe generation via in vitro transcription Preparing PCR products for in vitro transcription templates. Designing PCR primers with phage transcription promoter sequences in their 5′ ends. Note: The promoter sequence added to 5’end of the sense-strand PCR primer will be used for transcribing the sense probe, and the promoter sequence added to 5’end of the antisense PCR primer will be used for synthesizing the anti-sense probe1-3. We…

Discussion

The methods described in this protocol have been adapted from a number of different sources and optimized for whole mount E8.5-E11.5 day old mouse embryos. Methods for whole mount in situ hybridization of vertebrate embryos first appeared in the early 1990s ^2,5-12. This protocol was adapted primarily from methods developed for Xenopus embryos ^7,8 as well as mouse ^2,11. In our protocol a great deal of emphasis is placed on careful quality assessment of the probe. Careful atten…

Materials

Name	Type	Company	Catalogue number	Comments
Digoxigenin-11-uridine-5′-triphosphate	Reagent	Roche	11209256910
Ribonucleoside Triphosphate Set	Reagent	Roche	11277057001
Quick Spin Columns for radiolabeled RNA purification	Supply	Roche	11274015001
T7 RNA polymerase	Reagent	Roche	10881767001
SP6 RNA polymerase	Reagent	Roche	10810274001
T3 RNA polymerase	Reagent	Roche	11031163001
CTP, [α-32P]- 800Ci/mmol 10mCi/ml , 250 μCi	Reagent	Perkin Elmer	BLU008X250UC
DNase I recombinant, RNase-free	Reagent	Roche	4716728001
Urea,Colorless-to-white Crystals or Crystalline Powder	Reagent	Fisher	BP169-500
Gel loading buffer	Reagent	Ambion	AM8547
Hybond-N+, Amersham	Supply	GE Healthcare	RPN82B
UV Stratalinker 2400	Equipment	Stratagene
Diethyl pyrocarbonate	Reagent	Sigma	D5758-100ML
Heparin sodium	Reagent	Acros	41121-0010
hydrogen peroxide 30% in water	Reagent	Fisher scientific	BP2633-500
Gluteraldehyde, 8% EM grade	Reagent	Polysciences	0/710	Use with caution according to manufacture’s instructions
Blocking reagent	Reagent	Roche	11096176001	Make into 5% stock and store at -20° C
Proteinase K	Reagent	Roche	3115852001
Rnase A	Reagent	Roche	10109142001	Make as 10 mg/ml stock and store at -20° C.
Rnase T1	Reagent	Roche	10109193001
Ultrapure Formamide	Reagent	Invitrogen	15515-026
Levamisole hydrochloride	Reagent	ICN Biomedicals. Inc	155228
Ribonucleic acid from torula yeast,Type VI	Reagent	Sigma	R6625	phenol/chloroform extracted several times and precipitated, resuspended in DEPC-dH2O and stored at -20° C
Anti-Digoxigenin-AP Fab fragments	Reagent	Roche	11093274910
BM Purple AP Substrate, precipitating. Ready-to-use solution.	Reagent	Roche	11 442 074 001
4mL, Clear, Closed Top, Storage Vial Convenience Kit	Supply	National scientific	B7800-2
Shake N Bake hybridization oven	Equipment	Boekel Scientific	136400
Biopsy Sure-Tek	Supply	Fisherbrand	15-200-402C
Paraplast Plus tissue embedding medium	Reagent	Fisherbrand	23-021-400
Peel-A-Way disposable plastic tissue embedding molds	Supply	Polysciences	18646A
Colorfrost Plus Microscope slides	Supply	Fisherbrand	12-550-17
Nuclear Fast Red	Reagent	Sigma	N8002
Cytoseal 60	Reagent	Richard-Allan Scientific	8310-16