Name: Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos (Video) | JoVE Journal
Uploaded: 2013-01-30T13:00:00.000+00:00
Duration: 9 min 57 s
Description: Learn whole-mount RNA FISH for analyzing gene expression and localization in Drosophila melanogaster embryos. This optimized protocol enables high-resolution visualization of RNA distribution during embryogenesis.

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5 minutes

Work conducted in the L&eacute;cuyer laboratory is supported by funding from the National Sciences and Engineering Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR) and the Fonds de Recherche en Sant&eacute; du Qu&eacute;bec (FRSQ). Fabio Alexis Lefebvre and Ga&euml;l Moquin-Beaudry are supported by NSERC undergraduate research studentships, while Carole Iampietro is supported by the Angelo Pizzagalli postdoctoral fellowship.

1. RNA Probe Preparation
Overview: The following section describes the steps required to make Digoxigenin (Dig)-labeled RNA probes suitable for FISH. The first step involves cloning or PCR amplifying a sequence corresponding to the transcribed region of a gene of interest that will be used to generate a sequence-specific probe. This can be achieved by first cloning the gene segment into a plasmid in which the multiple cloning site is flanked by bacteriophage promoter elements (T7, T3 or Sp6), which can then serve as a template for PCR using flanking primers that incorporate the promoter sequences, thus generating a linear PCR product with different promoter sequences at each extremity (Figure 1A). Alternatively, to avoid the cloning step, one can also perform PCR amplification from genomic DNA or cDNA using oligonucleotides that include T7, T3 or Sp6 sequences at their 5' extremities (e.g. T7: 5'-TAATACGACTCACTATAGGGAGA-3'; T3: 5'-AATTAACCCTCACTAAAGGGAGA-3'; Sp6: 5'-ATTTAGGTGACACTATAGAAGAG-3'). The linear PCR products are then used as templates to make Dig-labeled sense or antisense RNA probes by run-off in vitro transcription with the appropriate polymerase (Figure 1B). When making probes for specific genes, we recommend preparing both sense and antisense RNA probes using distinct polymerases, which will be helpful for assessing FISH signal specificity (i.e. unless the gene of interest is transcribed in the antisense orientation, the sense RNA probe will reveal the level of background signal). In all of the following steps, one should take great care to avoid potential degradation by contaminating ribonucleases (RNAses) by first cleaning all bench areas and equipment with 70% ethanol or commercial RNAse decontamination solutions and by using RNAse-free supplies (e.g. water, tips, microcentrifuge tubes).
Materials
<ul>
 <li>1.5 ml microcentrifuge tubes, (Abgene, Rochester, NY, USA Cat. No 0900)</li>
 <li>Gel-extraction kits (QIAGEN, Mississauga, ON, Canada; Cat. No. 28706).</li>
 <li>RNAse free water (Wisent, Inc. Cat. No. 809-115-CL)</li>
 <li>RNA polymerases (T7, T3, or SP6). (20 U/ &mu;l, Fermentas Biosciences. Cat. No. EP0101, EP0111, EP0131).</li>
 <li>RNAse-OUT Ribonuclease inhibitors (40 U/ &mu;l), (Invitrogen, Burlington ON. Canada.Cat. No. 10777-019).</li>
 <li>Digoxigenin (DIG) nucleotide mixes (DIG-11-UTP, Roche Applied Biosciences, Laval, QC, Canada. Cat. No.11209256910).</li>
</ul>
Equipment
<ul>
 <li>Table top micro-centrifuge</li>
 <li>Gel-electrophoresis apparatus</li>
</ul>
<ol>
 <li>PCR amplify gene-specific sequence from genomic DNA, cDNA, or plasmid DNA to generate a linear PCR product flanked by bacteriophage T7, T3 or Sp6 promoter sequences. Follow the manufacturer's recommendations for PCR conditions.</li>
 <li>Verify the quality and size of the PCR product by agarose gel electrophoresis. Excise and purify PCR product using a standard gel-extraction kit according to the manufactures recommendations and elute the product in 50 &mu;l of buffer.</li>
 <li>Precipitate the PCR product by adding 5 &mu;l of 3M sodium acetate (pH 5.2) and 150 &mu;l of ice cold 100% ethanol, and place the tubes at -70 &deg;C overnight. Centrifuge tubes at 13,000 x g for 10 min at 4 &deg;C and wash the pellet with 70% ethanol. Spin again, remove all traces of ethanol and air dry the pellet. Resuspend in 25-50 &mu;l of RNAse-free water.</li>
 <li>Transcribe Dig-labeled probe using 200-500 ng purified PCR product in a 20 &mu;l reaction as follows: 2 &mu;l of 10X T7 transcription buffer (Invitrogen), 1 &mu;l (20 U/ &mu;l) of RNAse inhibitor, 2 &mu;l Dig-NTP mix, and 2 &mu;l T7 RNA polymerase (20 U/ &mu;l). Bring the final volume to 20 &mu;l with RNAse-free water. Incubate the transcription reaction at 37 &deg;C for 3-4 hr.</li>
 <li>Adjust transcription mix to 50 &mu;l with RNAse-free water and precipitate the Dig-labeled RNA probe as described in Step 4. Resuspend the washed RNA pellet in 100 &mu;l of RNAse-free water. Store the resuspended samples at -70 &deg;C.</li>
 <li>Quantify the probe using a nanodrop spectrophotometer. To verify the probe quality, run a 1-2 &mu;l sample on a 1-2% agarose gel stained with ethidium bromide.</li>
</ol>
2. Collection and Fixation of Drosophila Embryos
Overview: This section describes steps for harvesting and processing staged Drosophila embryo. Depending on the number of embryos needed, flies can be maintained in population cages of various sizes. The following steps are for collections performed using 900 cm3 cylindrical cages using 100mm apple juice collection plates. Proper fixation requires the removal of two protective layers surrounding the embryo: the outer chorion and the inner vitelline membrane 13. Once harvested, the embryos are first bathed in a 50% bleach solution to remove the chorion, then they are mixed in a biphasic solution containing heptane (permeabilizes the vitelline membrane), and PBS containing 3.7% formaldehyde. The vitelline membrane is then cracked and removed by transferring the embryos into a biphasic mixture of heptane and methanol. Proper embryo fixation and removal of the vitelline membrane is critical for the success of the downstream FISH procedure.
Materials
<ul>
 <li>Apple-juice agar plates (40% apple juice, 4% sucrose, 3.5% agar, 0.3% methyl paraben) with dime-sized yeast paste (baker's yeast mixed with water to butter consistency)</li>
 <li>Powdered paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA; Cat. No. 19208)</li>
 <li>Heptane</li>
 <li>Methanol</li>
 <li>3% Bleach solution</li>
 <li>20 ml glass scintillation vials (Thermo Fisher Scientific, Ottawa, ON, Canada; Cat. No. 03-337-15).</li>
</ul>
Equipment
<ul>
 <li>Population cages</li>
 <li>Collection baskets (made using a Nitex mesh and the top portion of a cut 50 ml polypropylene tube in which a hole has been cut in the lid).</li>
 <li>Small paint brush</li>
 <li>Table top vortex</li>
</ul>
<ol>
 <li>Pre-clear well-fed population cages with a fresh apple juice/yeast plate for 1 hr at 25 &deg;C.</li>
 <li>Add a new apple juice/yeast plate to the cage to harvest early stage embryos. Incubate the cage for 4 hr at 25 &deg;C (note that collection times can be adjusted depending on the desired stages).</li>
 <li>Prepare a fresh 37% formaldehyde solution by combining 3.7 g paraformaldehyde powder, 70 &mu;l of 2N KOH, and 7.3 ml of milliQ water in a glass scintillation vial under a chemical hood. Boil the solution until the paraformaldehyde powder is completely dissolved, then cool to room temperature and filter into new scintillation vial.</li>
 <li>Prepare a biphasic fixation solution by combining 2.25 ml of 1X PBS with 250 ml of 37% formaldehyde in a scintillation vial, then adding 7.5 ml of heptane.</li>
 <li>Harvest the apple juice plate from the cage and collect the embryos by adding a bit of room temperature tap water and delicately brushing the embryos off the plate with the paint brush. Transfer the resuspended embryos into a basket and rinse with lukewarm tap water.</li>
 <li>Dechorionate embryos for 90 sec by bathing the collection baskets in bleach solution, then rinse thoroughly with lukewarm tap water (until bleach smell is gone).</li>
 <li>Disassemble the collection basket, collect embryos gently using a paintbrush and transfer them into the biphasic fixation solution. The embryos will accumulate at the interface between the PBS and heptane layers.</li>
 <li>Seal scintillation vials and fasten to a vortex mixer. Shake for 20 min on lowest setting.</li>
 <li>Remove and discard most of the lower PBS and upper heptane phases using a Pasteur pipette. Aspirate the remaining PBS/heptane/embryos mixture into the pipette. In a dropwise fashion, discard the lower PBS phase from the pipette, taking care not to eliminate the embryos. Deposit the embryos into a 1.5 ml tube containing a biphasic solution of 500 &mu;l heptane (upper phase) and 500 &mu;l methanol (lower phase).</li>
 <li>Shake tubes vigorously by hand for 30-45 sec to crack the vitelline membranes. Once cracked, the embryos will sink into the methanol.</li>
 <li>Discard the upper heptane phase and uncracked embryos at the heptane/methanol interphase, and add 500 &mu;l of methanol. Shake for 5 sec.</li>
 <li>Wash 3 times with methanol and store the embryos at -20 &deg;C (at this point embryos can be stored for weeks/months).</li>
</ol>
3. Post-fixation Processing, Hybridization and Post-hybridization Washes
Overview: This section details the processing steps for embryo permeabilization prior to FISH, pre-hybridization, hybridization with the RNA probes and post-hybridization washes. For the initial permeabilization steps the embryos are handled as a pool in a single tube (steps 1-9 below, aiming for 10-15 &mu;l of settled embryos/sample), but they are aliquoted into individual wells of a 96-well PCR plate before proceeding with the pre-hybridization step (step 10 below). This helps to ensure even treatment of all embryos in the initial phases of the experiment. When setting up a FISH experiment, it is important to include negative controls to determine the level of background signal in individual experiments. For this, we recommend including a 'no-probe' sample and, as noted in section 1, a sample hybridized with the 'sense' RNA probe, which will typically give a signal comparable to the 'no-probe' condition.
Materials:
<ul>
 <li>Methanol</li>
 <li>Phosphate buffered saline with 0.1% Tween-20 (PBT)</li>
 <li>Proteinase-K 1 mg/ml stock solution (Sigma Aldrich, Oakville, ON, Canada. Catalog No. P2308)</li>
 <li>Glycine (20 mg/ml stock solution)</li>
 <li>Hybridization solution: 50% Formamide, 5X SSC, 0.1% Tween-20, 100 g/ml of sheared salmon sperm DNA, 100 &mu;g/ml Heparin.</li>
 <li>0.2 ml halfskirted 96-well PCR plates, Abgene, Rochester, NY, USA Cat. No 0900)</li>
</ul>
Equipment
<ul>
 <li>Hybridization oven, digital heating block or water bath adjustable to 56 &deg;C, 80 &deg;C or 100 &deg;C, or a PCR machine.</li>
 <li>Multichannel pipettors (recommended to simplify washing steps)</li>
 <li>Nutating mixer</li>
</ul>
<ol>
 <li>Rinse the embryos in methanol, then in a 1:1 mixture of methanol:PBT, and then twice in PBT.</li>
 <li>Fix the embryos for 20 min in 1 ml of PBT containing 3.7% formaldehyde (freshly prepared as described in step 11) with constant rocking on a nutator.</li>
 <li>Wash embryos for 3 times for 2 min in PBT while rocking.</li>
 <li>Prepare a solution of 3 &mu;g/ml of proteinase K (PK) diluted in PBT and add 500 &mu;l of solution to samples. Incubate samples for 10 min at room temperature without shaking. Mix embryos every 2 min by jetting with a pipette or by gently inverting the tubes.</li>
 <li>Transfer embryos samples on ice for 1 hr.</li>
 <li>Move samples to room temperature and remove PK solution. Wash 2 times for 2 min with 1 ml of PBT + 2 mg/ml glycine.</li>
 <li>Fix samples 20 min in 1 ml of PBT + 3.7% formaldehyde with rocking. Rinse embryos 5 times in PBT.</li>
 <li>Rinse embryos with 1 ml of a 1:1 mixture of PBT and Hyb solution. Replace with 0.5-1 ml of 100% Hyb solution (at this step the embryos can be stored for several days/weeks at -20 &deg;C).</li>
 <li>Aliquot embryos into individual wells of a 96-well plate (aim for ~10-15 &mu;l of settled embryos/well, take care to use wide aperture tips to avoid damaging the embryos).</li>
 <li>Boil 100 &mu;l/sample of Hyb solution for 5 min and then cool on ice for 5 min. This solution is used for sample pre-hybridization (Pre-Hyb).</li>
 <li>Add 100 &mu;l/sample of Pre-Hyb solution and incubate for at least 2 hr at 56 &deg;C.</li>
 <li>For each sample, dilute ~100 ng of probe in 100 &mu;l of Hyb solution. Heat at 80 &deg;C for 3 min, then cool on ice for 5 min (heated probes can be kept on ice for several hours).</li>
 <li>Remove the Pre-Hyb solution from the embryos and replace with the RNA probe solution.</li>
 <li>Hybridize at 56 &deg;C for 12-16 hr.</li>
 <li>Pre-warm the following wash solutions at 56 &deg;C: (A) 200 &mu;l/sample of Hyb solution; (B) 100 &mu;l/sample of a 3:1 mix of Hyb:PBT; (C) 100 &mu;l/sample of a 1:1 mix of Hyb:PBT; (D) 100 &mu;l/sample of a 1:3 mix of Hyb:PBT; (E) 400 &mu;l/sample of PBT.</li>
 <li>Rinse samples with 100 &mu;l of wash solution A, then perform the wash step with 100 &mu;l/sample of solutions A-D at 56 &deg;C for 15 min each. Then, wash samples 4 times for 5 min with 100 &mu;l/sample of solution E at 56 &deg;C.</li>
 <li>Cool samples to room temperature.</li>
</ol>
4. Probe Detection
Overview: This section describes the antibodies and detection reagents used to detect and visualize the distribution of the RNA probe signal following hybridization. These steps are outlined schematically in Figure 2. Here we only present the standard detection reagents that we use for robust signal amplification, but there exists a variety commercially available reagents that can be used as alternatives, especially when performing double-FISH experiments to co-detect different RNAs simultaneously for protein-RNA double staining. Examples of results obtained with this protocol are displayed in the mRNA expression/localization pattern mosaic in Figure 3.
Materials
<ul>
 <li>HRP-conjugated mouse monoclonal anti-DIG (Jackson ImmunoResearch Laboratories Inc. Cat. No. 200-032-156)</li>
 <li>Biotin-conjugated mouse monoclonal anti-DIG (Jackson ImmunoResearch Laboratories Inc. Cat. No. 200-062-156)</li>
 <li>Streptavidin-HRP conjugate (Molecular Probes, Eugene OR, USA, Cat. No.S991)</li>
 <li>Cy3-tyramide conjugates (Perkin Elmer Life Sciences, Boston, MA, USA, Cat. No. SAT704A)</li>
 <li>DABCO mounting solution: In a 50 ml tube, dilute 1.25 grams of DABCO (1,4-diazabicyclo [2.2.2] octane; Sigma D-2522) in 15 ml of 1X PBS, then add 35 ml of glycerol and mix until fully homogeneous. By premixing with PBS, the DABCO powder is dissolved very quickly. Store mounting solution at -20 &deg;C.</li>
 <li>Glass slides, coverslips</li>
</ul>
Equipment
<ul>
 <li>Nutating mixer</li>
 <li>Multichannel pipettor</li>
 <li>Fluorescence microscope</li>
</ul>
<ol>
 <li>Prepare PBTB solution, containing PBT + 1% non-fat dry milk (usually 50-100 ml is sufficient for all the detection reagent incubation and washing steps).</li>
 <li>Block samples for 10 min at room temp in 100 &mu;l PBTB/sample while rocking on nutator.</li>
 <li>Remove blocking solution from embryos and incubate samples for 1.5-2 hr at room temperature with 100 &mu;l/sample the monoclonal biotin anti-DIG antibody solution (diluted 1/400 from stock into PBTB) with rocking.</li>
 <li>Wash samples 6 times for 5 min each with 100 &mu;l PBTB/sample with rocking.</li>
 <li>Incubate 1.5 hr at room temperature with 75 ml/sample of streptavidin-HRP solution (diluted 1/100 from stock in PBTB, 10 &mu;g/ml final concentration) with rocking.</li>
 <li>Wash samples 6 times for 5 min each 100 &mu;l PBTB/sample with rocking (for the DNA counterstaining, dilute DAPI to 1X working concentration in PBTB and use this solution in the first wash step).</li>
 <li>Rinse embryos with PBT, then wash 2 times for 5 min with PBS.</li>
 <li>Incubate samples for 2 hr at room temperature on the nutator with 50 &mu;l/sample of Cy3-tyramide solution (diluted 1/50 in Tyramide amplification buffer). From this step on, make sure to keep samples in a light shielded receptacle.</li>
 <li>Wash samples 6 times for 5 min each with 100 &mu;l PBS/sample on nutator.</li>
 <li>Remove PBS and add 100-125 &mu;l/sample of mounting solution containing 70% glycerol and 2.5% (DABCO). Store samples at 4 &deg;C. These samples can be stored for several years.</li>
 <li>Using a wide-bore tip, resuspend the embryos by gently pipetting the samples and transfer 25 &mu;l of embryos onto a glass slide, then place a coverslip over the embryos and seal onto the slide with fingernail polish.</li>
 <li>Analyze embryo samples using a fluorescence microscope.</li>
</ol>

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No conflicts of interest declared.

For probe synthesis steps, we typically generate run-off antisense RNA probes by in vitro transcription from full-length Drosophila cDNAs amplified from plasmids found in the Drosophila Gene Collection (DGC), a resource detailed at the following website: <a href="http://www.fruitfly.org/DGC/index.html" target="_blank">http://www.fruitfly.org/DGC/index.html</a> 14,15. This approach has been used extensively in large scale ISH studies aimed at mapping gene expression and mRNA localizat...

<table><tbody><tr><td>T7, T3 or SP6 RNA Polymerase </td><td>Fermentas Life Sciences</td><td>EP0101,EP0111,EP0131</td><td>Kits contain reaction buffer. </td></tr><tr><td>DIG RNA Labeling Mix </td><td>Roche Applied Science</td><td>11 277 073 910</td><td></td></tr><tr><td>RNAguard </td><td>Amersham Biosciences</td><td>27-0816-01</td><td></td></tr><tr><td>3M sodium acetate </td><td></td><td></td><td></td></tr><tr><td>Cold 100% ethanol. </td><td></td><td></td><td></td></tr><tr><td>Cold 70% ethanol. </td><td></td><td></td><td></td></tr><tr><td>Chlorine bleach solution diluted 1:1 with water. </td><td></td><td></td><td></td></tr><tr><td>Heptane </td><td></td><td></td><td></td></tr><tr><td>Methanol </td><td></td><td></td><td></td></tr><tr><td>proteinase K </td><td>Sigma Aldrich Oakville, ON, Canada</td><td>Catalog No. P2308</td><td></td></tr><tr><td>40% formaldehyde solution, freshly prepared </td><td></td><td></td><td></td></tr><tr><td>PBS-Tween solution (PBT) </td><td></td><td>1xPBS, 0.1% Tween-20</td><td></td></tr><tr><td>Glycine solution </td><td></td><td>2 mg/ ml glycine in PBT</td><td></td></tr><tr><td>HRP-conjugated mouse monoclonal anti-DIG </td><td>Jackson ImmunoResearch Laboratories Inc</td><td>200-032- 156</td><td>(1/400 dilution of a 1 mg/ml stock solution in PBTB </td></tr><tr><td>HRP-conjugated sheep monoclonal anti-DIG </td><td>Roche Applied Science, Laval, QC</td><td>1 207 733</td><td>1/500 dilution of stock solution in PBTB </td></tr><tr><td>Biotin-conjugated mouse monoclonal anti-DIG </td><td>Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA</td><td>200-062-156</td><td>(1/400 dilution of a 1 mg/ml stock solution in PBTB </td></tr><tr><td>Streptavidin-HRP conjugate </td><td>Molecular Probes, Eugene OR, USA</td><td>S991</td><td>(1/100 dilution of a 1 &amp;mu;g/ml stock </td></tr></tbody></table>

whole mount rna fluorescent in situ hybridization of drosophila embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

When performed successfully, this procedure offers a strikingly enhanced level of detail in the spatio-temporal analysis of gene expression and mRNA localization dynamics during early Drosophila embryogenesis. Indeed, as illustrated in Figure 3A for the classical pair-rule gene runt (run), one can use this protocol to observe gene expression events via the detection of nascent transcript foci in groups of expressing nuclei. In addition, as shown in the embryo mosaic in <s...

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

This procedure describes an optimized fish method to assess expression and localization features of RNA in whole mount Drosophila embryos. This is accomplished by first synthesizing a labeled RNA probe, complimentary to an Mr NA or non-coating RNA of interest by in vitro transcription of an appropriately selected DNA template. In parallel fly population cages are used to harvest, fix and perme embryos of the desired developmental stage.After several post fixation steps, the embryos are hybridized with the labeled antisense RNA probe. Finally, following extensive washing of the samples, the hybridized probe is detected by immuno labeling with a specific antibody and fluorochrome conjugated detection reagents, thus providing a sensitive readout of the target RNA distribution. Ultimately, the results yield highly resolved fluorescent signals that are analyzed by standard fluorescence microscopy in order to document the spatial and temporal expression features of the target RNA during drosophila embryogenesis.The main advantage of this technique over existing methods such as Northern blot or R-T-P-C-R is one can assess the RNA expression within intact tissue specimen. While this method is intended for the analysis of RNA expression properties in early drosophila embryos, it can also be employed for the analysis of embryos at later developmental stages of tissue dissected from larvae or adult flies. An important aspect of our procedure is upscaling in sample number that we can achieve by performing fish in PCR place, which substantially increases throughput of the approach.The members of the lab demonstrating this procedure include undergraduate research resistance, graduate students, and post-doctoral fellows. Begin with well-fed population cages. Replace the plate with an apple juice yeast plate and leave the flies for an hour at 25 degrees Celsius.Then replace the apple juice yeast plate to harvest early stage embryos. Incubate the cage at 25 degrees Celsius and wait until the desired developmental stage of the embryos has been reached while waiting in a chemical hood, mix the chemicals to make 37%formaldehyde in a glass inhalation vial. Boiled the solution until the PFA powder is completely dissolved.Then cool it to room temperature and filter it into a new scintillation vial. Next, prepare a biphasic fixation solution. Combine PBS with 37%formaldehyde in a scintillation vial, and then add heptane when ready.Harvest the embryos from the apple juice plate with room temperature, tap water, and delicate sweeping with a fine paintbrush. Transfer the resuspended embryos into a basket and rinse them with lukewarm tap water. Now dec coate the embryos for 90 seconds by bathing the basket in a fresh 3%bleach solution.Then remove the bleach with lukewarm tap water until the bleach smell is gone. To collect the embryos, disassemble the collection basket and gently transfer the embryos into the biphasic fixation solution. Using the paintbrush, the embryos will accumulate at the interface between the PBS and heptane layers.Now seal the scintillation vials, fasten them to a vortex mixer and shake them for 20 minutes on the lowest setting. After the shaking, discard most of the lower PBS and upper heane phases using a pasture pipette. Then aspirate the remaining mixture into the pipette and carefully drop out the lower phase without losing the embryos.Deposit the embryos into a 1.5 milliliter tube containing a biphasic solution of 500 microliters of heptane and 500 microliters of methanol. Shake the tube vigorously for 30 to 45 seconds to crack the patella membranes. Once cracked, the embryos will sink into the methanol.Discard the upper phase and uncracked embryos and add one milliliter of methanol. Now briefly shake the tube for five seconds and wash the embryos three times with methanol. After the washes, the embryos can be stored for several months at minus 20 degrees Celsius.Begin by adding 500 microliters of PK to each of embryos and incubate the samples for 10 minutes at room temperature without shaking. Take great care to damage the samples by over digesting them with protein a k, or through sloppy manipulation. During the PK incubation, mix the embryos every two minutes by jetting them with a pipette.After 10 minutes, put the embryos on ice for an hour. After an hour, remove the PK solution at room temperature. Wash the embryos twice with glycine in PBT for two minutes.Now, fix the samples for 20 minutes in one milliliter of 3.7%formaldehyde in PBT with rocking. Wash out the formaldehyde with five rinses in PBT. Then rinse the embryos with one milliliter of equal volumes PBT and HI solution, and replace the mixture with 0.5 to one milliliter of pure solution.At this point, the embryos can be stored for several days to weeks at minus 20 degrees Celsius. In a 96 well plate aliquot 10 to 15 microliters of settled embryos per well. Use a wide bore tip to avoid damaging the embryos.Now prepare the pre hybridization solution by boiling 100 microliters of hype solution per sample. Well for five minutes, cool the Pre-Hype solution on ice for five minutes, and then add 100 microliters of it to each plate well, which contains the embryo samples. Retrieve the prepared RNA probe quantified by a NanoDrop spectrophotometer.Also, to verify the probe's quality, run one to two microliters of it on an agro gel. Now for each sample, dilute about 100 nanograms of probe in 100 microliters of HI solution. It is vital to work under RNAs free conditions.When handling the probe. Heat the solution to 80 degrees Celsius for three minutes. Then cool it on ice for at least five minutes.It can be kept cooled for several hours. Retrieve the plate and remove the Pre-Hype solution from the embryos by aspiration. Then add 100 microliters of hype solution with the RNA probe to each.Well hybridize the probe at 56 degrees Celsius for 12 to 16 hours the next day. A series of five solutions are prewarm at 56 degrees Celsius. The first is pure hype solution, and the last is pure PBT.The other three are dilution of HI into PBT. Once warmed quickly rinse the samples once in pure hype solution. Next, perform three 15 minute washes, beginning with the least diluted hype solution followed by each of the successive dilutions.Lastly, wash the samples four times for five minutes in pure PBT. Then bring the samples to room temperature. Now proceed with antibody applications to detect the probe to improve sample mixing.During these steps, the plate can be placed in a small box that is attachable to a mutating mixer using the described protocol, the classic pair rule gene runt was analyzed at distinct stages of embryogenesis. Fish analysis revealed how the initial broad expression in the mid portion of the embryo at embryonic stage four is refined into a segmented stripe pattern. In later stages, fish was performed using various diggen and labeled antisense, RNA probes hybridized to zero to four hour old drosophila embryos.The hybridized probes were detected by sequential incubations with a biotinylated antigen and antibody strep avid in HRP and Ceramide. The samples were mounted and analyzed by fluorescence microscopy. A broad mosaic of fish results were observed in these stage four to seven embryos While attempting this procedure, it's important to use precautions at various steps such as protecting your RNA probes and degradation by working under RNAs free conditions and using RNAs free supplies.Once this basic is mastered, additional steps can be added to the method to do multi labeling experience in order to visualize different RNAs or specific protein markers in the same sample. After watching this video, you'll have a good idea of how to perform fluorescent in C two hybridization in order to document the expression or subcellular localization dynamics of your favorite RNA during Drosophila embryogenesis Happy Fishing.

whole-mount-rna-fluorescent-situ-hybridization-drosophila

Research

JoVE Journal

Biology

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.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

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 of E8.5 to E11.5 Mouse Embryos

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Learn whole-mount RNA FISH for analyzing gene expression and localization in Drosophila melanogaster embryos. This optimized protocol enables high-resolution visualization of RNA distribution during embryogenesis.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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