Name: Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos (Video) | JoVE Journal
Uploaded: 2011-10-10T12:00:00.000+00:00
Duration: 13 min 54 s
Description: Learn whole mount in situ hybridization for gene expression studies in E8.5-E11.5 mouse embryos. This protocol enables reproducible visualization of developmental patterns using ribo probes and immunohistochemical detection.

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This work was supported by NIH grants R21MH082360 (BGC) and R01HD056315 (NRM) as well as the University of Georgia.

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<li>Parr, B. A., Shea, M. J., Vassileva, G., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+Wnt+genes+exhibit+discrete+domains+of+expression+in+the+early+embryonic+CNS+and+limb+buds%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.</a> Development. 119, 247-261 (1993).</li>
<li>Rosen, B., Beddington, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-mount+in+situ+hybridization+in+the+mouse+embryo%3A+gene+expression+in+three+dimensions%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions.</a> Trends Genet. 9, 162-167 (1993).</li>
<li>Quiring, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+expression+screening+by+automated+whole-mount+in+situ+hybridization%5BTitle%5D)+AND+%22Mech.+Dev%22%5BJournal%5D)">Large-scale expression screening by automated whole-mount in situ hybridization.</a> Mech. Dev. 121, 971-976 (2004).</li>
<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev. Biol. 231, 63-76 (2001).</li>
<li>Neidhardt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+screen+for+genes+controlling+mammalian+embryogenesis%2C+using+high-throughput+gene+expression+analysis+in+mouse+embryos%5BTitle%5D)+AND+%22Mech.+Dev%22%5BJournal%5D)">Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.</a> Mech. Dev. 98, 77-94 (2000).</li>
<li>Gordon, J., Bennett, A. R., Blackburn, C. C., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gcm2+and+Foxn1+mark+early+parathyroid-+and+thymus-specific+domains+in+the+developing+third+pharyngeal%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal.</a> 103, 141-143 (2001).</li>
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No conflicts of interest declared.

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...

<table><tbody><tr><td>Digoxigenin-11-uridine-5&amp;rsquo;-triphosphate</td><td>Reagent</td><td>Roche Group</td><td>11209256910</td><td></td></tr><tr><td>Ribonucleoside Triphosphate Set</td><td>Reagent</td><td>Roche Group</td><td>11277057001</td><td></td></tr><tr><td>Quick Spin Columns for radiolabeled RNA purification</td><td>Supply</td><td>Roche Group</td><td>11274015001</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>Reagent</td><td>Roche Group</td><td>10881767001</td><td></td></tr><tr><td>SP6 RNA polymerase</td><td>Reagent</td><td>Roche Group</td><td>10810274001</td><td></td></tr><tr><td>T3 RNA polymerase</td><td>Reagent</td><td>Roche Group</td><td>11031163001</td><td></td></tr><tr><td>CTP, [&amp;#945;-32P]- 800Ci/mmol 10mCi/ml , 250 &amp;mu;Ci</td><td>Reagent</td><td>PerkinElmer, Inc.</td><td>BLU008X250UC</td><td></td></tr><tr><td>DNase I recombinant, RNase-free</td><td>Reagent</td><td>Roche Group</td><td>4716728001</td><td></td></tr><tr><td>Urea,Colorless-to-white Crystals or Crystalline Powder</td><td>Reagent</td><td>Fisher Scientific</td><td>BP169-500</td><td></td></tr><tr><td>Gel loading buffer</td><td>Reagent</td><td>Ambion</td><td>AM8547</td><td></td></tr><tr><td>Hybond-N+, Amersham</td><td>Supply</td><td>GE Healthcare</td><td>RPN82B</td><td></td></tr><tr><td>UV Stratalinker 2400</td><td>Equipment</td><td>Stratagene, Agilent Technologies</td><td></td><td></td></tr><tr><td>Diethyl pyrocarbonate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>D5758-100ML</td><td></td></tr><tr><td>Heparin sodium</td><td>Reagent</td><td>Acros Organics</td><td>41121-0010</td><td></td></tr><tr><td>hydrogen peroxide 30% in water</td><td>Reagent</td><td>Fisher Scientific</td><td>BP2633-500</td><td></td></tr><tr><td>Gluteraldehyde, 8% EM grade</td><td>Reagent</td><td>Polysciences, Inc.</td><td>0/710</td><td>Use with caution according to manufacture&amp;rsquo;s instructions</td></tr><tr><td>Blocking reagent</td><td>Reagent</td><td>Roche Group</td><td>11096176001</td><td>Make into 5% stock and store at -20&amp;#x00B0; C</td></tr><tr><td>Proteinase K</td><td>Reagent</td><td>Roche Group</td><td>3115852001</td><td></td></tr><tr><td>Rnase A </td><td>Reagent</td><td>Roche Group</td><td>10109142001</td><td>Make as 10 mg/ml stock and store at -20&amp;#x00B0; C.</td></tr><tr><td>Rnase T1</td><td>Reagent</td><td>Roche Group</td><td>10109193001</td><td></td></tr><tr><td>Ultrapure Formamide</td><td>Reagent</td><td>Invitrogen</td><td>15515-026</td><td></td></tr><tr><td>Levamisole hydrochloride</td><td>Reagent</td><td>ICN Biomedicals</td><td>155228</td><td></td></tr><tr><td>Ribonucleic acid from torula yeast,Type VI</td><td>Reagent</td><td>Sigma-Aldrich</td><td>R6625</td><td>phenol/chloroform extracted several times and precipitated, resuspended in DEPC-dH2O and stored at -20&amp;#x00B0; C</td></tr><tr><td>Anti-Digoxigenin-AP Fab fragments</td><td>Reagent</td><td>Roche Group</td><td>11093274910</td><td></td></tr><tr><td>BM Purple AP Substrate, precipitating. Ready-to-use solution.</td><td>Reagent</td><td>Roche Group</td><td>11 442 074 001</td><td></td></tr><tr><td>4mL, Clear, Closed Top, Storage Vial Convenience Kit</td><td>Supply</td><td>National Scientific Company</td><td>B7800-2</td><td></td></tr><tr><td>Shake N Bake hybridization oven</td><td>Equipment</td><td>Boekel Scientific</td><td>136400</td><td></td></tr><tr><td>Biopsy Sure-Tek</td><td>Supply </td><td>Fisher Scientific</td><td>15-200-402C</td><td></td></tr><tr><td>Paraplast Plus tissue embedding medium</td><td>Reagent</td><td>Fisher Scientific</td><td>23-021-400</td><td></td></tr><tr><td>Peel-A-Way disposable plastic tissue embedding molds</td><td>Supply</td><td>Polysciences, Inc.</td><td>18646A</td><td></td></tr><tr><td>Colorfrost Plus Microscope slides</td><td>Supply</td><td>Fisher Scientific</td><td>12-550-17</td><td></td></tr><tr><td>Nuclear Fast Red</td><td>Reagent</td><td>Sigma-Aldrich</td><td>N8002</td><td></td></tr><tr><td>Cytoseal 60</td><td>Reagent</td><td>Richard-Allan Scientific</td><td>8310-16</td><td></td></tr></tbody></table>

whole mount in situ hybridization of e8.5 to e11.5 mouse embryos

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

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.

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.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

The overall goal of this procedure is to define gene expression patterns in whole mouse embryos. This is accomplished by first designing and generating a specific ribo probe for the gene of interest. Next, the embryos are collected and treated for hybridization.Then the labeled probe is hybridized to the embryos. The final step is to visualize the hybridized probe by immunohistochemical detection. Ultimately, results can be obtained that show gene expression patterns in whole embryos or embryonic sections.After imaging with a stereos, dissecting microscope, or a regular brightfield microscope, Generally individuals new to this method will struggle because the institute hybridization procedures are lengthy and technically demanding, there are multiple important steps that collectively contribute to the quality of the final result. First, generate template DNA by amplifying the sequence of interest using gene specific PCR primers designed with farge transcription promoter sequences on the five prime ends. Then purify the PCR product and include a proteinase K digestion step according to the written protocol.Then set up an in vitro transcription reaction and incubator optimal temperature for two hours. After DNA's digestion of the DNA template, purify the ribo probe using column purification, estimate probe yield by using a NanoDrop or microplate absorption reader. Then adjust the probe concentration to 100 nanograms per microliter according to the estimated yield.Then check the quality of the probe by denaturing acrylamide gel electrophoresis as outlined in the written protocol and by measuring the specific activity of the RIBA probe, a good probe should yield a single discrete band on the gel with minimal smear to measure specific activity, perform serial dilution of the ribo probe to obtain a range of dilutions from 10 to the minus two to 10 to the minus five. Then pipette one microliter of each dilution onto an 82 millimeter diameter piece of high bond n plus membrane, along with one microliter of an unlabeled DNA sample as a control. Crosslink the filter immediately in a UV crosslinker at 125 millijoules after crosslinking.Add 10 milliliters of 1%blocking reagent in one times TN buffer incubate room temperature for 30 minutes after blocking is complete. Remove the blocking solution and wash the filter for 15 minutes at room temperature in 10 milliliters of one times TN buffer while washing the membrane. Prepare a one to 5, 000 dilution of anti doxygen in FAB fragment in 10 milliliters of one times TN buffer after washing.Incubate the membrane in this solution for 30 minutes Following incubation, wash the membrane twice for 15 minutes each in 10 milliliters of one times TN buffer. After the final wash, aspirate the wash solution from the Petri dish and replace with five milliliters of BM purple substrate. Incubate the membrane in this solution for 30 minutes.After 30 minutes have elapsed, check the membrane for the presence of a visible signal emitted from the spot corresponding to the 10 to the minus four dilution. If the signal is visible, stop the color reaction by washing with five times TE twice for five minutes each time. If no signal is visible after 30 minutes, the probe should be discarded.Collect mouse embryos in ice cold PBST. Then sort the embryos according to genotype into four milliliter screw capped files containing PBST. Fix the embryos with 4%PARALDEHYDE in PBST unless otherwise specified.All incubation and wash steps are done with solutions filled to the lower level of the vial neck, which is about four milliliters in volume. Leave the embryos overnight at four degrees Celsius after fixation. Aspirate the 4%paraldehyde in PBST with a glass pasta pipette and replace with PBST.Place the vial on ice for five minutes to wash. Repeat this process to wash the tissue for a second time. Next, aspirate the PBST and dehydrate the embryos stepwise through a methanol in PBST series into 100%methanol.Note that the pipette tip is kept on the opposite side of the vial to avoid damage to the embryos if desired, the embryos can be stored at minus 20 degrees Celsius in 100%methanol for up to eight months and maybe longer until use. Transfer the embryos to a dish, puncture the heart and head of E 10.5 and older embryos with a microdissection knife while the embryos are still in methanol. This allows wash solutions to enter the brain ventricles and heart chambers and reduces background staining.Then rehydrate the embryos by successive washes in a methanol in PBST series. Finishing with two washes for five minutes each in 100%PBST bleach the embryos by preparing a solution of four parts PBST to one part 30%hydrogen peroxide and incubating the embryos in this solution for one hour on ice. During the incubation, place the freshly prepared hybridization buffer at 65 degrees Celsius to prewarm.Wash the embryos with PBST twice for five minutes each. During the final wash, make a 20 milligrams per milliliter proteinase K stock with DEPC water. Then serially dilute the stock to make 10 micrograms per milliliter.Proteinase K in PBST aspirate the final wash solution and replace with one milliliter of the 10 microgram per milliliter proteinase K solution. Place the vial vertically in a tube rack within a 25 to degrees Celsius water bath and incubate for 10 to 30 minutes, depending on the size of the embryos. Make a note of the start time as optimal and replicable.Incubation time is critical to enable penetration of the ribo probe without over digestion of the embryo. To stop the protease K digestion aspirate the proteinase K solution 30 seconds before the incubation time has elapsed. Then when the incubation time elapses add freshly made two milligrams per milliliter glycine in PBST.Let's stand for five minutes at room temperature. Then repeat the glycine wash for another five minutes. Then wash twice in PBST for five minutes each.Next refix the proteinase K treated embryos by immersing the embryos in 4%PARALDEHYDE in PBST supplemented with 0.2%glutaraldehyde for 20 minutes. Then wash the embryos in PBST three times for five minutes each. After the last wash, aspirate the PBST and replace with one milliliter of prewarm hybridization buffer.Once the embryos have settled, aspirate the hybridization buffer and replace with one milliliter of fresh prewarm hybridization.Buffer. Incubate the vials vertically in a 65 degrees Celsius oven with a shaking platform for one hour. Prepare hybridization buffer containing 0.5 micrograms per milliliter of labeled ribo.Probe for embryos up to E 8.5 or 0.1 to one microgram per milliliter of probe for embryos past E 8.5. Following the incubation, replace the pre hybridization solution with 0.4 milliliters of this solution and hybridize overnight at 65 degrees Celsius. The next day, check the quality of the embryos.If the embryos have been overtreated with proteinase K, they may stick to the size of the vial, will appear extremely transparent or fall apart. Embryos that appear this way should be discarded as processing them further will not result in interpretable data. Replace the hybridization buffer with four milliliters of solution.One at 70 degrees Celsius. Maintain this temperature and twice for 30 minutes each for E 8.5 embryos or three times for 30 minutes per wash. For E 9.5 and older embryos during the last wash, prepare four milliliters of 50%solution, one 50%solution two for each vial and prewarm to 70 degrees Celsius after the last wash.Incubate the embryos in this solution for 10 minutes, then wash three times for five minutes per wash with four milliliters of solution. Two at room temperature with rocking after washing. Replace the solution with fresh solution.Two containing 100 micrograms per milliliter RNAs A and 100 units per milliliter RNAs T one incubate at 37 degrees Celsius twice for 30 minutes per wash. Next washing solution three at 65 degrees Celsius with rocking twice for 30 minutes each wash for E 8.5, three times for 30 minutes per wash for E 9.5 and older. Finally, wash three times for five minutes per wash with one times TBST.Then proceed to antibody detection. Aspirate the final TBST wash from the vial and replace with 0.5 milliliters of one times TBST plus 10%heat treated sheep serum. Place the vial vertically on a platform shaker at room temperature and incubate with rocking for at least two and a half hours to block non-specific antibody binding sites.After the incubation time has elapsed, aspirate the blocking solution and replace with fresh one times TBST plus 10%serum containing a one to 2000 to one to 5, 000 dilution of subtracted alkaline phosphatase, conjugated sheep antigen, and FAB fragments antibody and incubate overnight at four degrees Celsius. Wash three times for five minutes each with one times TBST. Then five to six times for one hour reach at room temperature and finally overnight at four degrees Celsius to remove excess antibody.The next day. Wash the embryos twice for 20 minutes per wash with freshly made alkaline phosphatase buffer. This is an important procedure to reduce background.Replace the last wash with one milliliter of prewarm BM purple AP substrate. Incubate at room temperature while protected from light and watch for signal periodically via dissecting microscope signal. For abundant messenger.RNA should be visible within 15 minutes. When the signal is clearly visible or if background staining begins to be a problem, stop the reaction by washing twice for five minutes per wash with one times PBST plus five millimolar EDTA capture images to document the staining pattern of the whole mount embryo. Once this has been done, return the embryos to a vial of BM purple substrate and incubate for 16 to 24 hours at room temperature until the external surface of the embryo becomes dark blue to allow the stain to penetrate throughout the embryo.Then transfer the embryos to 4%parmal hide in PBST to refix for several hours or overnight at four degrees Celsius. This final fixation step is crucial for long-term preservation of the staining pattern. Finally, proceed to paraffin, embedding and sectioning according to the procedures outlined in the written protocol to reveal this staining.This image shows a successful hole mount in C two hybridization of an E 10.5 embryo using a gata three specific ribo probe. Here is an image showing an E 11.5 embryo that has been successfully labeled with a Fox G one specific ribo probe. This image shows high background and weak signal from a failed in C two hybridization with a partially degraded gata three probe illustrating the importance of performing careful quality control of probes to ensure high quality results.Here is an image of another unsuccessful procedure. In this case, the procedure was performed without puncturing the head resulting in a high level of background staining in the ventricles as indicated by the arrow heads. After watching this video, you should have a good understanding of how to perform high quality Institute hybridization to define gene expression patterns on mouse embryos for spatial details and the temporal or genotype comparisons.

whole-mount-in-situ-hybridization-of-e85-to-e115-mouse-embryos

Research

JoVE Journal

Biology

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Whole Mount in situ Hybridization of Early Chick Embryos

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Learn whole mount in situ hybridization for gene expression studies in E8.5-E11.5 mouse embryos. This protocol enables reproducible visualization of developmental patterns using ribo probes and immunohistochemical detection.

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

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