Funding support for this project was provided by a grant to HMK by the Canadian Institutes of Health Research (grant MOP 133473).

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Rev Genet. 10, 57-63 (2009).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98, 81-85 (1989).</li><li>Hughes, S. C., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+labeling+with+fluorescence+in+situ+hybridization+in+Drosophila+whole-mount+embryos.">Double labeling with fluorescence in situ hybridization in Drosophila whole-mount embryos.</a> Biotechniques. 24, 530-532 (1998).</li><li>Lecuyer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li><li>Kosman, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Raap, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-sensitive+FISH+using+peroxidase-mediated+deposition+of+biotin-+or+fluorochrome+tyramides.">Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides.</a> Human Mol Genet. 4, 529-534 (1995).</li><li>Lecuyer, E., Parthasarathy, N., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+protocols+in+Drosophila+embryos+and+tissues.">Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.</a> Methods Mol Biol. 420, 289-302 (2008).</li><li>Wilk, R., Murthy, S. U. M., Yan, H., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols Essential Laboratory Techniques. , 1-9 (2010).</li><li>Holt, C. E., Bullock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+mRNA+Localization+in+Animal+Cells+and+Why+It+Matters.">Subcellular mRNA Localization in Animal Cells and Why It Matters.</a> Science. 326, 1212-1216 (2009).</li><li>Martin, K. C., Ephrussi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+Localization:+Gene+Expression+in+the+Spatial+Dimension.">mRNA Localization: Gene Expression in the Spatial Dimension.</a> Cell. , 719-730 (2009).</li><li>Wilk, R., Hu, J., Blotsky, D., Krause, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+and+pervasive+subcellular+distributions+for+both+coding+and+long+noncoding+RNAs.">Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.</a> Genes Dev. 30, 594-609 (2016).</li><li>Zhou, X. L., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximo-distal+specialization+of+epithelial+transport+processes+within+the+Xenopus+pronephric+kidney+tubules.">Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules.</a> Dev Biol. 271, 322-338 (2004).</li></ol>

Authors have no competing financial interests.

The described protocol provides a highly reproducible, sensitive, and economical method for the detection of most RNAs in fixed Drosophila embryos or tissues. Although slightly more complicated than the more traditionally used alkaline phosphatase method of probe detection, the resolution obtained by FISH is far greater and the sensitivity is comparable or improved 7,8. By using whole or partial microtiter plates, the protocols can be used for large-scale or limited analyses of genes of interest. It should be noted that the probes generated may detect all or multiple transcript splice forms unless probes are more specifically designed (i.e., single exons). Although we have tested probes as small as 200 nucleotides with reasonable success, smaller probes tend to be less effective and may be less specific. Clearly, this protocol is not appropriate for the detection of small introns, exons, or processed microRNAs.
Although this approach uses an enzymatic step to boost the strength of signals, signal intensity appears to be proportional to target gene expression in a relatively linear and broad range of expression levels. At higher magnification, the signal is seen as puncta or speckles within cells and tissues. For relatively rare transcripts only a few tiny puncta are seen. As transcript abundance increases, these grow in number and size. As with single molecule FISH (smFISH), single small puncta may also correspond to single RNAs and should also be readily quantified using imaging and informatics software. Comparisons of published images obtained using smFISH with images that we have curated for the same targets suggest that the overall, sensitivity and resolution of the two methods are relatively similar, although this should be tested by more rigorous comparisons. Given the much higher expense of smFISH, the method provided here should give similar results at a fraction of the cost. However, if the goal is to detect small transcripts, or distinct parts of transcripts, smFISH is recommended.
Due to the smaller size of some FISH probes, this method may also be better at penetrating tissues that are large or have significant barriers. However, our use of higher detergent levels and tissue fixation and permeabilization tweaks appear to have solved this problem. Finally, although developed for Drosophila tissues, the high detergent buffers described here for dissected tissues should also work for Drosophila embryos as well as most other organisms and tissues.

New genome-wide RNA detection methods such as RNA-seq have greatly expanded our knowledge of when and where genes are expressed, and at what levels1. However, these provide relatively poor temporal and spatial resolution. RNA in situ hybridization allows the spatial distribution of transcripts to be visually observed in fixed tissues, thus revealing the details of cellular and subcellular localization2. Using fluorescent in situ hybridization (FISH) allows even more spatial resolution as it allows the use of powerful microscopy techniques, such as confocal and light sheet microscopy3. Fluorescent markers such as DAPI can also be used to establish subcellular relationships4. FISH also facilitates the observation of multiple RNAs and proteins simultaneously with clear indications of overlap at the subcellular level. Unique reagents and steps required for double-labeling can be found in the following references3,5.
The procedures described here utilize 96-well microtiter plates for all steps, including cDNA template production (colony PCR), RNA probe production (using T7, T3 or SP6 polymerase-dependent run-off transcription), in situ hybridization, and fluorescent signal development. Sufficient numbers of embryos or tissues are added to each well of the 96-well plate to allow evaluation of consistency and variability. Each well then receives a different probe. After signal development, embryos or tissues from each well are arrayed on individual microscope slides for microscopic analysis.
The use of tyramide signal amplification (TSA) for probe detection produces robust signals with exceptional subcellular resolution 5,6,7,8. The subcellular localization of RNAs is an important regulatory mechanism 9 that appears to occur with virtually all RNAs 4,10,11. These analyses have also shown that many transcripts that are not detected by RNA-seq are readily detected by FISH 8. The adaptation of RNA in situ hybridization to 96-well plates allows analysis of up to 96 genes of interest at a time (more if additional plates are used), making analysis of large datasets possible. By cutting the plates into smaller sections, the method is also easily adapted to just a few samples. The protocol provided is appropriate for analysis of RNA distributions in most types of tissues. Although not shown, it is adaptable to non-Drosophila tissues as well.

<table><tbody><tr><td>15 ml Polypropylene conical centrifuge tubes</td><td>Frogga Bio</td><td>TB15-500</td><td>Certified DNase and RNase free</td></tr><tr><td>2x PCR Master Mix with dye(Ambiogene)</td><td>Biomart (Canada)</td><td>141106-1K</td><td>Certified DNase and RNase free</td></tr><tr><td>50 ml Polypropylene conical centrifuge tubes</td><td>Frogga Bio</td><td>TB50-500</td><td>Certified DNase and RNase free</td></tr><tr><td>96-well filteration plate (1.2 um, hydrophilic PVDF membrane</td><td>EMD MILLIPORE</td><td>MSBVS1210</td><td>Used to remove liguid from bottom of the plate with floting tissues</td></tr><tr><td>96-well PCR plate (200 &amp;mu;l)</td><td>Denville</td><td>C18096-10</td><td></td></tr><tr><td>Acrodisc syringe filter (0.2 u)</td><td>PALL</td><td>PN4612</td><td></td></tr><tr><td>Acrodisc syringe filter (0.45 u)</td><td>PALL</td><td>PN4614</td><td></td></tr><tr><td>Agarose</td><td>Bioshop</td><td>AGA002.500</td><td></td></tr><tr><td>AXYGEN 96-well assay storage plates</td><td>UltiDent Scientific</td><td>24-P96-450V-C</td><td>To store bacterial glycerol stocks</td></tr><tr><td>AXYGEN Aluminum Plate Seals</td><td>UltiDent Scientific</td><td>24-PCR-AS-200</td><td>To seal plates</td></tr><tr><td>AXYGEN Wide-bore tips (200 ul)</td><td>UltiDent Scientific</td><td>24-T205-WB-C</td><td>To transfer samples</td></tr><tr><td>Biotin-SP (long spacer) IgG Fraction Monoclonal Mouse Anti-Digoxin</td><td>Jackson ImmunoResearch Lab</td><td>200062156</td><td>Primary antibody for in situ (stock conc. 1 mg/ml, working conc. 2.5 &amp;mu;g/ml or 1/400 dilution.</td></tr><tr><td>BRAND 8-channel manifold (autoclavable)</td><td>BrandTech Scientific Inc.</td><td>704526</td><td>To remove liquids from the top of 96-well PCR plates</td></tr><tr><td>Chloramphenicol</td><td>Sigma-aldrich</td><td>C1919</td><td>Stock concentration 34 mg/ml dissolved and 0.2&amp;mu;m filtered in 100 % EtOH (use as 1/1000)</td></tr><tr><td>Cyanine 3 Tyramide (Cy3-TSA)</td><td>Home made</td><td></td><td>Could be ordered from Perkin-Elmer Cat# SAT704A001EA</td></tr><tr><td>DABCO (1,4-Diazabicyclo[2.2.2]octane)</td><td>Sigma-aldrich</td><td>D2522</td><td>Antifade reagent in mounting media</td></tr><tr><td>Digoxigenin-11-uridine-5&amp;rsquo;-triphosphate</td><td>Roche</td><td>11-209-256-910</td><td></td></tr><tr><td>Embryo collection cage</td><td>Home made</td><td>N/A</td><td>Size: 75 cm X 75 cm X 75 cm made with Polymethyl methacrylate</td></tr><tr><td>Eppendorf centrifuge 5804</td><td>Eppendorf</td><td>Model 5804</td><td>Centrifuge for 96-well plates on A-Z-DWP rotor</td></tr><tr><td>Formamide</td><td>Bioshop</td><td>FOR001.500</td><td></td></tr><tr><td>Glycerol</td><td>Bioshop</td><td>GLY002.4</td><td></td></tr><tr><td>Glycine</td><td>Bioshop</td><td>GLN001.500</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H4784-250MG</td><td>Stock: 50 mg/ml. Working concentration 1 mg/ml in Hybridization solution</td></tr><tr><td>Lab Armor Beads</td><td>DiaMed</td><td>LAA42370-001</td><td>Fill in heating unit</td></tr><tr><td>Labnet Gyro M Mini Nutating Mixer</td><td>Fisher Scientific</td><td>50-998-347</td><td>Sample mixing</td></tr><tr><td>Mesh - 100 micons</td><td>FlyStuff</td><td>57-103</td><td>For collecting embryos</td></tr><tr><td>Mesh - 800 microns</td><td>SEFAR - Nytex</td><td>06-780/53</td><td>For catching loose tissue / may be used for special tube in figure 4.</td></tr><tr><td>Micro Cover Glasses</td><td>VWR</td><td>48366-067</td><td>Size: 22 X 22 mm</td></tr><tr><td>Micro slides,frstd</td><td>VWR</td><td>48312-003</td><td>Size: 25 X 75 mm</td></tr><tr><td>Multi-Well Plate Vacuum Manifold</td><td>Pall Corporation</td><td>P/N 5017</td><td>Remove liquid from bottom of 96-well filter plate</td></tr><tr><td>Paraformaldehyd (PFA)</td><td>Bioshop</td><td>PAR070.500</td><td>Fixation</td></tr><tr><td>PCR machine</td><td>Bio-Rad</td><td>Model: DNA Engine PTC-200</td><td>96-well plate PCR and probe denature</td></tr><tr><td>Picric acid solution</td><td>Sigma-aldrich</td><td>80456</td><td>For tissue fixation I solution</td></tr><tr><td>Potassium Chloride (KCL)</td><td>Bioshop</td><td>POC308</td><td></td></tr><tr><td>PP lid for 96-well plates, 100/case</td><td>UltiDent Scientific</td><td>24-P-LID-PP</td><td>Lid for storing plates</td></tr><tr><td>Progene disposable Reagent Reservoirs (50 ml, sterile)</td><td>UltiDent Scientific</td><td>825-1-50-5S</td><td>Liquid handling</td></tr><tr><td>Progene Pierceable Aluminum Foil</td><td>UltiDent Scientific</td><td>87-CFILM-AL</td><td>Seal 96-well plate, DNase and RNase free</td></tr><tr><td>Proteinase K</td><td>Sigma-aldrich</td><td>P2308- 5MG</td><td>Embryo permeabilization</td></tr><tr><td>RiboLock Ribonuclease Inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td>40 Unit/ul</td></tr><tr><td>Ribonucleoside Triphosphate (NTP) set</td><td>Roche</td><td>11277057001</td><td>RNA probe sythesis</td></tr><tr><td>RNase free water</td><td>GIBCO</td><td>10977015-500ml</td><td>RNA probe sythesis</td></tr><tr><td>RNaseZap</td><td>Ambion</td><td>AM9780</td><td>Remove RNase contamination</td></tr><tr><td>Single stranded DNA from salmon testes</td><td>Sigma-aldrich</td><td>D9156</td><td>For hybridization solution</td></tr><tr><td>Skim milk (non fat power)</td><td>Bioshop</td><td>SKI400.500</td><td>Working concentration is 1 % in 1 X PBT or PBTT as a blocking reagent</td></tr><tr><td>Sodium Acetate (NaOAc) 3 M, pH=5.2 (RNase free)</td><td>Bioshop</td><td>SAA333</td><td>Working concentration is 10 % (v/v) for DNA or RNA precipitation</td></tr><tr><td>SP6 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0131</td><td>20 Unit/ul</td></tr><tr><td>Stainless Steel Shot for Sand Bath</td><td>VWR</td><td>13259-274.</td><td>To fill the heating unit</td></tr><tr><td>Stereomicroscope &amp;amp; gooseneck light source</td><td>Leica</td><td>Model: MZ6</td><td>Tissue dissection and mounting</td></tr><tr><td>Streptavidin, horseradish peroxidase (HRP) conjugate</td><td>Molecular Probes</td><td>S911</td><td>Secondary antibody stock: 1 mg/ml, use 1/1000 dilution (1 &amp;mu;g/ml) for in situ.</td></tr><tr><td>T3 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0101</td><td>20 Unit/ul</td></tr><tr><td>T7 RNA Polymerase</td><td>Thermo Scientific</td><td>EP0111</td><td>20 Unit/ul</td></tr><tr><td>Tip station (10 ul)</td><td>Axygen</td><td>T-300-STK-S</td><td>Certified DNase and RNase free</td></tr><tr><td>Tip station (200 ul)</td><td>Sorbio</td><td>27770T</td><td>Certified DNase and RNase free</td></tr><tr><td>Tips ( 1ml)</td><td>Sorbio</td><td>10200</td><td>Certified DNase and RNase free</td></tr><tr><td>TRITON X-100</td><td>Bioshop</td><td>TRX506</td><td>Working concentration is 0.3% in 1 X PBT for tissue in situ</td></tr><tr><td>Tween 20</td><td>Bioshop</td><td>TWN510</td><td>Working concentration is 0.1% in 1 X PBT for embryo in situ</td></tr><tr><td>Whatman qualitative filter paper, Grade 3</td><td>Sigma-aldrich</td><td>WHA1003917</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Preparation&lt;/strong&gt;</td></tr><tr><td>DABCO (1,4-Diazabicyclo[2.2.2]octane) glycerol anti-fade mounting media</td><td></td><td></td><td>DABCO (1.25 g), Glycerol (35 ml or v/v 70 %) and 1 X PBS (15 ml, or v/v 30 %). Keep dark and rock in cold room. store at -20 &amp;deg;C</td></tr><tr><td>1 X PBT (1L)</td><td></td><td></td><td>1 X PBS with 1 ml Tween 20 (0.1 %)</td></tr><tr><td>1 X PBTT (1L)</td><td></td><td></td><td>1 X PBS with 1 ml Tween 20 (0.1 %) and 3 ml of Triton-X-100 (0.3 %)</td></tr><tr><td>1 X PBTB (1L)</td><td></td><td></td><td>1 % Skim milk in 1 X PBT</td></tr><tr><td>1 X PBTTB (1L)</td><td></td><td></td><td>1 % Skim milk in 1 X PBTT</td></tr></tbody></table>

high resolution fluorescent in situ hybridization in drosophila embryos and tissues using tyramide signal amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

Figure 6 shows examples of results obtained using this procedure. The top 3 panels show examples of early Drosophila embryos (Figure 6, panels A-C), the next 3 panels show examples of late Drosophila embryos with signals in different tissues (amnioserosa, muscles and central nervous system, respectively (Figure 6, panels D-F). Next are examples from 3rd instar larval tissues (Figure 6, panels G-J). Panels K and L show representative images from adult gonads (ovary and testis, respectively). Hundreds of images have been uploaded and annotated in our searchable 'fluorescent in situ fruit fly database' (FlyFISH (http://fly-fish.ccbr.utoronto.ca)).
<img alt="Workflow diagram for RNA FISH in Drosophila; PCR, in situ hybridization, imaging steps shown." class="xfigimg" src="/files/ftp_upload/56281/56281fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Outline of Fluorescent in situ hybridization (FISH) protocol and key equipment. (A) PCR amplification of cDNA. (B) In vitro transcription to generate gene-specific antisense DIG- labeled RNA probes in a 96-well plate (photo of the plate shown in b). (C) and (D): Adults (female: male ratio 2:1. 300-400 flies/bottle) flies are allowed to mate for 3-4 days. Embryos from an overnight collection on standard cornmeal food at 25 °C are transferred to a well ventilated plastic box (1 L of cornmeal food in a container sized: 8" X 8" X 3" LWH) or to new bottles keeping a proper larval density. For tissue collection, active yeast powder should be sprinkled on top of the food at 24, 48 h after egg laying (AEL). (E to H): Fluorescent in situ hybridization experiments performed over 3 consecutive days. For embryos or tissues that do not float, use a 96-well PCR plate as shown in photograph b with 8-channel aspirator (photograph c). For tissues that float, like most larval tissues, use a filter-bottomed plate (photograph in d) and remove liquid from bottom with a manifold aspirator using low pressure (photograph in e). <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gel electrophoresis result; RNA probes, DNA template separation; molecular weight markers." class="xfigimg" src="/files/ftp_upload/56281/56281fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Determination of RNA probe yield. Newly synthesized antisense RNA probes observed on a 1 % agarose gel (5 µL/lane). The yield can be categorized (based on the intensity of the band) as 'low yield' in lanes 3 and 10, 'typical yield' in lanes 1, 4, 5, 6 and 8, or 'high yield' in lanes 2, 7, 9, 11, and 12. For samples with 'typical yields', use 10-15 µL of probe diluted in 100 µL of hybridization solution for each in situ experiment. Dilute samples with 'high yields' of probe with additional hybridization solution according to the band intensity relative to a 'typical probe' intensity. Aim to have roughly equal final probe concentrations in each well. The arrow points to one of the 'RNA probes', the higher band on each lane is the original DNA template. <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Drosophila embryo preparation diagram: collection, dechorionation, rinsing, fixation, devitellinization." class="xfigimg" src="/files/ftp_upload/56281/56281fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Major steps for a large-scale embryo collection and fixation. <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Centrifuge tube with filter setup, protein purification process, laboratory equipment." class="xfigimg" src="/files/ftp_upload/56281/56281fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Homemade tube for removing liquid from larval or floating tissues. Some larval and adult tissues float in solution during fixation. This simple tool consists of a nylon mesh held by a 200 µL cut tip, followed by a 1 mL tip as an adaptor to connect to an aspirator. Liquid can be safely removed without losing any tissue. <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tyramide signal amplification diagram: streptavidin-HRP binds probe, enhances fluorescence." class="xfigimg" src="/files/ftp_upload/56281/56281fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Tyramide Signal Amplification. (1) DIG-labeled antisense RNA probe hybridizes with endogenous sense RNA. (2) Immuno-affinity binding between biotin-conjugated primary antibody and DIG-UTP. (3) HRP conjugated streptavidin binds biotin. (4) HRP catalyzes the cyanine 3-tyramide and hydrogen peroxide reaction to form short-lived cyanine 3-tyramide radicals. (5) Cyanine 3-tyramide radicals bind to tyrosine residues on nearby proteins. <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescent microscopy images, gene expression analysis, Drosophila tissue sections, cellular localization." class="xfigimg" src="/files/ftp_upload/56281/56281fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Representative in situ hybridization images. Each panel shows an example of a different RNA (shown in Cyan) and nuclei stained with DAPI (shown in red). (A-C) Examples of early Drosophila embryos. (D, F) Examples of late Drosophila embryos. (G-J) Examples of 3rd instar Drosophila larval tissues (malphigian tubules, midgut, salivary gland and muscle respectively). (K) Adult ovary. (L) Adult testis. Each panel displays the name of the gene that the probe is hybridizing to. Scale bars = 10 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56281/56281fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification at JoVE.com

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

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

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

high-resolution-fluorescent-situ-hybridization-drosophila-embryos

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12.9K Views.  University of Toronto. Het beschreven RNA in situ hybridisatie protocol staat toe de opsporing van RNA in hele Drosophila embryo's of ontleed weefsels. Met behulp van 96-wells microtiterplaat platen en tyramide signaal versterking, kunnen afschriften worden gedetecteerd op hoge resolutie, gevoeligheid en doorvoer, en tegen een relatief lage prijs.

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

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

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

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

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

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.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Developmental Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged molecules within complex biological samples. While traditional confocal microscopes allow one to resolve two adjacent fluorescent point sources located a few hundred nanometers apart, observing the finer details of subcellular biology requires the ability to resolve signals in the order of tens of nanometers. Numerous hardware-based methods for super-resolution microscopy have been developed to allow researchers to sidestep such resolution limits, although these methods require specialized microscopes that are not available to all researchers. An alternative method for increasing resolving power is to isotropically enlarge the sample itself through a process known as expansion microscopy (ExM), which was first described by the Boyden group in 2015. ExM is not a type of microscopy per se but is rather a method for swelling a sample while preserving the relative spatial organization of its constituent molecules. The expanded sample can then be observed at an effectively increased resolution using a traditional confocal microscope. Here, we describe a protocol for implementing ExM in whole-mount Drosophila embryos, which is used to examine the localization of Par-3, myosin II, and mitochondria within the surface epithelial cells. This protocol yields an approximately four-fold increase in sample size, allowing for the detection of subcellular details that are not visible with conventional confocal microscopy. As proof of principle, an anti-GFP antibody is used to distinguish distinct pools of myosin-GFP between adjacent cell cortices, and fluorescently labeled streptavidin is used to detect endogenous biotinylated molecules to reveal the fine details of the mitochondrial network architecture. This protocol utilizes common antibodies and reagents for fluorescence labeling, and it should be compatible with many existing immunofluorescence protocols.

Using Expansion Microscopy to Physically Enlarge Whole-Mount Drosophila Embryos for Super-Resolution Imaging

Here, a protocol for the implementation of expansion microscopy in early Drosophila embryos to achieve super-resolution imaging using a conventional laser-scanning confocal microscope is presented.

The workhorse of developmental biology is the confocal microscope, which allows researchers to determine the three-dimensional localization of tagged ...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

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.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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 ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

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..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

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

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