We would like to thank Athanasios Papangelis, for critical comments on the manuscript. FM is supported by the Biotechnology and Biological Sciences Research Council (BBSRC; BB/M009424/1). IP is supported by the American Heart Association Scientist Development Grant (17SDG33060002).

Mouse heart tissue samples for this study were obtained in compliance with the relevant laws and institutional guidelines and were approved by Yale University Institutional Animal Care and Use Committee.
1. Solution Preparation
<ol>
	<li>Prepare RNase free ddH2O, by treating 5 L of ddH2O with 5 mL of 0.1% diethylpyrocarbonate (DEPC) overnight (O/N), at room temperature (RT). Autoclave (121&#160;&#176;C) to deactivate the DEPC. Use DEPC-treated ddH2O for the preparation of downstream solutions as indicated. 
	CAUTION: DEPC is a known carcinogen, only handle in the fume hood.</li>
	<li>Prepare 1x Phosphate Buffered Saline (PBS) solution by dissolving 5 PBS tablets in 1 L of DEPC-treated ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare 0.1% non-ionic detergent/PBS (PBST) by diluting 1 mL of non-ionic detergent per 1 L of 1x PBS.</li>
	<li>Prepare 90%, 70%, 50% and 30% ethanol in DEPC-treated ddH2O.</li>
	<li>Prepare a 10 mg/mL stock solution of Proteinase K (ProK) in DEPC-treated ddH2O. Aliquot and store at -20&#160;&#176;C. Prepare a 20 &#956;g/mL working solution of ProK by diluting 100 &#956;L of ProK stock solution in 50 mL of DEPC-treated ddH2O. Make fresh before use.</li>
	<li>Prepare 4% paraformaldehyde (PFA) by adding 2 g of PFA in 50 mL of 1x PBS. Heat at 65&#160;&#176;C with occasional shaking, until dissolved. Aliquot and store at -20&#160;&#176;C. 
	Caution: PFA is a known carcinogen, only handle in the fume hood.</li>
	<li>Prepare a 3 M NaCl solution by adding 87.8 g to 500 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare a 1 M MgCl2 solution by adding 20.3 g to 100 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Prepare 1 M Tris pH 8.0 by dissolving 121.1 g in 800 mL of ddH2O. Adjust the pH to 8.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121&#160;&#176;C). Prepare a working solution of 50 mM Tris pH 8.0 by diluting 2.5 mL of 1 M Tris pH 8.0 in 47.5 mL of ddH2O.</li>
	<li>Prepare 1 M Tris pH 9.5 by dissolving 60.6 g in 400 mL of ddH2O. Adjust the pH to 9.5 with a few drops of 1 N HCl, bring the volume to 500 mL and autoclave (121&#160;&#176;C).</li>
	<li>Prepare 0.13 M 1-Methylimidazole pH 8.0 by diluting 1.6 mL of 1-Methylimidazole to 130 mL of DEPC-treated ddH2O. Adjust the pH to 8.0 with approximately 450 &#181;L of 12 N HCl. Add 16 mL of 3 M NaCl and bring the volume to 160 mL. Make fresh before use. 
	Caution: 1-Methylimidazole is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.</li>
	<li>Prepare 0.16 M N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC) by diluting 176 &#181;L of EDC to 10 mL of 0.13 M 1-Methylimidazole. Adjust the pH to 8.0 with approximately 100 &#181;L of 12 N HCl. Make fresh before use. 
	CAUTION: EDC is harmful to mucous membranes, eyes, and skin, only handle in the fume hood.</li>
	<li>Prepare 1% H2O2 by diluting 1.5 mL of 30% H2O2 in 50 mL of 1x PBS. Make fresh before use.</li>
	<li>Prepare 20x SSC pH 7.0 by dissolving 175.3 g of NaCl and 88.2 g of sodium citrate (Na3C6H5O7) in 800 mL of ddH2O. Adjust the pH to 7.0 with a few drops of 1 N HCl, bring the volume to 1 L and autoclave (121&#160;&#176;C).
	<ol>
		<li>Prepare a working solution of 2x SSC pH 7.0 by diluting 20 mL of 20x SSC pH 7.0 in 180 mL of ddH2O.</li>
		<li>Prepare a working solution of 1x SSC pH 7.0 by diluting 2.5 mL of 20x SSC pH 7.0 in 47.5 mL of ddH2O.</li>
		<li>Prepare a working solution of 0.2x SSC pH 7.0 by diluting 5 mL of 2x SSC pH 7.0 in 45 mL of ddH2O.</li>
	</ol>
	</li>
	<li>Prepare 1x hybridization solution by diluting 0.5 mL of 2x microRNA ISH buffer in 0.5 mL of DEPC-treated ddH2O. Make fresh before use.</li>
	<li>Prepare 200 mM stock solution of Levamisole by adding 250 mg to 5 mL of ddH2O. Aliquot and store at -20&#160;&#176;C.</li>
	<li>Prepare blocking solution for step 5 (10% sheep serum/1% BSA/0.2 mM Levamisol) by diluting 1 mL of sheep serum in 9 mL PBST, and adding 0.1 g of BSA. Add 10 &#956;L of Levamisole before using. Make fresh before use.</li>
	<li>Prepare blocking solution for step 6 (10% goat serum/1% BSA) by diluting 1 mL of goat serum in 9 mL of PBST, and adding 0.1 g of BSA. Store at 4&#160;&#176;C and use within a week.</li>
	<li>Prepare prestaining solution by diluting 3.3 mL of 3 M NaCl, 5 mL of 1 M MgCl2,10 mL of 1 M Tris pH 9.5, 100 &#181;L of Tween-20, 100 &#181;L of Levamisole in 81.5 mL of ddH2O. Make fresh before use.</li>
	<li>Use nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indoly phosphate (BCIP) tablets to prepare 20 mL of Alkaline Phosphatase (AP) substrate solution according to the manufacturer&#39;s instructions. Add 20 &#956;L of Levamisole. Make fresh before use and protect from light.</li>
	<li>Prepare KTBT solution by adding 4 g of Tris, 4.4 g of NaCl and 375 mg of KCl in 500 mL of ddH2O. Autoclave (121&#160;&#176;C).</li>
	<li>Optional: Prepare a DAPI working solution (1 &#956;g/mL) by diluting 50 &#956;L of DAPI stock solution (1 mg/mL) in 50 mL of 1x PBS. 
	NOTE: Standard solutions such as 3 M NaCl, 1 M MgCl2, 1 M Tris-HCl, nuclease-Free ddH2O, 1x PBS and 20x SSC can be alternatively purchased. For this protocol, 50 mL glass Coplin jars or 20 mL polypropylene slide mailer jars have been used.</li>
</ol>
2. Tissue Preparation
NOTE: The mouse heart sections used here were prepared by Yale Pathology Tissue Services, from Formalin-Fixed/Paraffin-embedded tissue, cut at 5 &#956;m and positioned on charged slides.
<ol>
	<li>Tissue rehydration.
	<ol>
		<li>Warm the slides at 60&#160;&#176;C for 10 min, in the hybridization oven until paraffin is visibly melting.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of commercially available clearing agent (see Table of Materials) for 10 min, twice.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of 100% ethanol for 2 min, twice, followed by 90% ethanol for 2 min, 70% ethanol for 2 min, twice, 50% ethanol for 2 min and 30% ethanol for 2 min.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of DEPC-treated ddH2O for 5 min.</li>
		<li>Incubate the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min.</li>
	</ol>
	</li>
	<li>Tissue de-proteinating.
	<ol>
		<li>Incubate slides in a glass Coplin jar containing 50 mL of 20 &#956;g/mL ProK working solution, at 37&#160;&#176;C for 15 min in the hybridization oven. 
		CAUTION: Under-digestion may result in poor or no signal development, while over-digestion may result in tissue degradation and development of background staining. Optimization may be required for this step (1&#8211;20 &#956;g/mL ProK, 5&#8211;20 min incubation, RT-37&#160;&#176;C).</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.</li>
	</ol>
	</li>
	<li>Tissue fixation.
	<ol>
		<li>Use a hydrophobic pen to draw a water repellent circle around the tissue on each slide.</li>
		<li>Apply 500 &#181;L of 4% PFA solution to the length of each slide and incubate slides horizontally for 10 min, at RT.</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS for 5 min, at RT, twice.</li>
		<li>Wash the slides in a glass Coplin jar containing 50 mL of 0.13 M 1-methylimidazole for 10 min, at RT, twice.</li>
		<li>Apply 500 &#181;L of 0.16 M EDC solution to the length of each slide and incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 50 mM Tris pH 8.0 at RT.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice. 
		NOTE: Both PFA and EDC fixation steps are required for miRNA crosslinking and should not be omitted.31</li>
	</ol>
	</li>
	<li>Endogenous peroxidase block.
	<ol>
		<li>Incubate slides in a glass Coplin jar containing 50 mL of 1% H2O2 for 30 min at RT.</li>
		<li>Rinse the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT, twice.</li>
	</ol>
	</li>
</ol>
3. Hybridization
<ol>
	<li>Preheat hybridization solution (500 &#181;L per slide) at 50&#160;&#176;C in the hybridization oven, until the target temperature, is reached (10&#8211;15 min).</li>
	<li>Prepare the hybridization chamber by placing 2 pieces of filter or tissue paper in the bottom of the chamber and wetting the paper with 50% formamide/1x SSC. 
	Caution: Formamide is harmful to mucous membranes, eyes and skin, only handle in the fume hood.</li>
	<li>Apply 200 &#181;L of hybridization solution to the length of each slide. Incubate slides horizontally for 1&#8211;3 h at 50&#160;&#176;C, in a humidified chamber.</li>
	<li>Prepare the probe solution by mixing 0.5 &#181;L stock probe (25 &#181;M) in 250 &#181;L ofhybridization solution (final concentration 50 nM) in a 1.5 mL tube. 
	NOTE: Different miRNAs are expressed at different levels, so optimization regarding the probe concentration may be required for this step (1&#8211;50 nM), to avoid no signal or high background development.</li>
	<li>Apply 250 &#181;L of probe solution to the length of each slide and place an RNase free coverslip on top. Avoid the formation of bubbles.</li>
	<li>Carefully seal the hybridization chamber with parafilm and incubate O/N at 50&#160;&#176;C. 
	NOTE: The hybridization temperature should be set approximately 30&#160;&#176;C below the RNA melting temperature (Tm) of each probe. Optimization may be required. Here, 50&#160;&#176;C is used as the hybridization/wash temperature for miRNA-182, adjust accordingly for different probes.</li>
</ol>
4. Stringency Washes
<ol>
	<li>Prewarm 200 mL of 2x SSC at 50&#160;&#176;C in the hybridization oven, until the target temperature is reached (20&#8211;40 min).</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at 50&#160;&#176;C for 5 min, or until the coverslips loosen.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 2x SSC at RT for 5 min, twice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 0.2x SSC at RT for 5 min.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min. 
	NOTE: Use of RNase-free conditions is no longer necessary since the RNA-RNA hybrids are considered stable.</li>
</ol>
5. DIG Antibody Detection
<ol>
	<li>Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.</li>
	<li>Apply 500 &#181;L of blocking solution to the length of each slide. Incubate slides horizontally for 30 min at RT, in a humidified chamber.</li>
	<li>Prepare the DIG antibody solution (1:1,000) by diluting 0.5 &#956;L of DIG antibody in 500 &#181;L ofblocking solution in a 1.5 mL tube. 
	CAUTION: Optimization may be required for this step (1:500&#8211;1:2,000).</li>
	<li>Apply 500 &#181;L of DIG antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of PBST at RT for 5 min, thrice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of prestaining solution at RT for 5 min, twice.</li>
	<li>Incubate slides in a polypropylene slide mailer jar containing 20 mL ofAP substrate solution at 37&#160;&#176;C, for 6&#8211;24 h, protected from light. 
	Caution: Color development should be checked every 2 h. Optimization may be required for this step.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of KTBT solution at RT for 5 min, twice.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min.</li>
</ol>
6. cTnT Antibody Detection
<ol>
	<li>Use a hydrophobic pen to re-draw a water repellent circle around the tissue on each slide if necessary.</li>
	<li>Apply 500 &#181;L of blocking solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Prepare the cTnT antibody solution (1:100) by diluting 2 &#956;L of cTnT antibody in 200 &#956;L ofblocking solution in a 1.5 mL tube.</li>
	<li>Apply 200 &#181;L of cTnT antibody solution to the length of each slide. Incubate slides horizontally O/N at 4&#160;&#176;C, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.</li>
	<li>Prepare the anti-rabbit-568 antibody solution (1:500) by diluting 0.5 &#956;L of anti-rabbit-568 antibody in 250 &#956;L ofblocking solution in a 1.5 mL tube.</li>
	<li>Apply 250 &#181;L of anti-rabbit-568 antibody solution to the length of each slide. Incubate slides horizontally for 1 h at RT, in a humidified chamber.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, thrice.</li>
	<li>Optional: Apply 250 &#181;L of DAPI working solution to the length of each slide, and incubate slides horizontally for 1 min at RT, protected from light.</li>
	<li>Wash the slides in a glass Coplin jar containing 50 mL of 1x PBS at RT for 5 min, twice.</li>
</ol>
7. Mounting and Imaging
<ol>
	<li>Mount the slides with 2&#8211;3 drops of mounting medium and a glass cover slip. Allow the slides to dry flat, O/N, at 4&#160;&#176;C, protected from light.</li>
	<li>Proceed to epifluorescent or confocal microscopy.</li>
</ol>

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<li>Burchfield, J. S., Xie, M., Hill, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathological+ventricular+remodeling%3A+mechanisms%3A+part+1+of+2%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Pathological ventricular remodeling: mechanisms: part 1 of 2.</a> Circulation. 128, 388-400 (2013).</li>
<li>Benjamin, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+Disease+and+Stroke+Statistics-2017+Update%3A+A+Report+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association.</a> Circulation. 135, e146-e603 (2017).</li>
<li>Pena, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miRNA+in+situ+hybridization+in+formaldehyde+and+EDC-fixed+tissues%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.</a> Nature Methods. 6, 139-141 (2009).</li>
</ol>

The authors have nothing to disclose.

miRNA transcript detection can be achieved through different techniques that vary in sensitivity, specificity and quantitative power. Here, we demonstrate the coupling of miRNA in situ hybridization with immunostaining and describe a protocol that allows for concurrent assessment of the expression levels of miRNA and protein molecules, on the same heart sections. We first show how to perform in situ hybridization of DIG-labeled LNA miRNA probes on paraffin embedded heart sections. Next, we describe how ...

micro-RNAs (miRNAs) are short (18&#8211;25 nucleotides), single-stranded, noncoding RNAs that function as negative regulators of gene expression at the post-transcriptional level by inhibiting messenger RNA (mRNA) translation and/or promoting mRNA degradation1. miRNAs are transcribed from introns or exons of coding or noncoding genes and are cleaved in the nucleus by DROSHA, to precursor miRNAs (pre-miRNAs), which are short stem-loop structures of 70 nucleotides2. Following cytoplasmic export, pre-miRNAs are further processed by DICER into mature miRNAs that span 18&#8211;25 nucleotides3,4. Subsequently, the RNA-induced silencing complex (RISC) incorporates these miRNAs as single-stranded RNAs, which allows for their binding to the 3&#39; untranslated region (3&#39;-UTR) of their target mRNAs to suppress their expression3,5.
Within the last three decades, since they were first identified, miRNAs have emerged to master&#160;regulators of gene expression, whose own expression levels are tightly controlled6. Roles for miRNAs have been described in organ development7,8,9,10,11,12, maintenance of homeostasis13,14, as well as disease contexts that include neurological15,16,17,18,19, cardiovascular20, autoimmune conditions21,22, cancers23,24, and others25. The increasing appreciation for the relevance of miRNA expression patterns has brought forward the need for reliable detection methods of miRNA transcripts. Such methods include Real Time PCR, microarrays, Northern Blotting, in situ hybridization and others, which vary in the sensitivity, specificity, and quantitative power, predominantly due to the fact that miRNA transcripts are comprised of short and highly homologous sequences6.
We recently reported an important role for miRNA-182 in the development of the myocardial hypertrophy26, a condition describing the structural adaptation of the heart in response to elevated hemodynamic demands27,28. Cardiac hypertrophy is characterized by the increase in the myocardial mass, which, if associated with maladaptive remodeling29, can lead to increased risk for heart failure, a condition accounting for 8.5% of all deaths attributable to cardiovascular disease30.
Here, we describe our protocol that combines in situ hybridization with a digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe and immunostaining for the concurrent detection of miRNA and protein molecules on mouse heart tissue sections, in our model of cardiac hypertrophy.

<table><tbody><tr><td>Diethylpyrocarbonate</td><td>Sigma Aldrich</td><td>D5758</td><td>DEPC</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td>PBS</td></tr><tr><td>Tween-20</td><td>American Bioanalytical</td><td>AB02038</td><td>non-ionic surfactant</td></tr><tr><td>Histoclear</td><td>National Diagnostics</td><td>HS-200</td><td></td></tr><tr><td>Proteinase K, recombinant, PCR Grade</td><td>Sigma Aldrich</td><td>3115879001</td><td>ProK</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>PFA</td></tr><tr><td>Sodium Chloride</td><td>ThermoFisher</td><td>S271</td><td>NaCl</td></tr><tr><td>Magnesium Chloride Hexahydrate</td><td>ThermoFisher</td><td>M33</td><td>MgCl&lt;sub&gt;2&lt;sub&gt;</td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>T6066</td><td></td></tr><tr><td>Hydrochloric Acid Solution, 1 N</td><td>ThermoFisher</td><td>SA48</td><td>HCl</td></tr><tr><td>Hydrochloric Acid Solution, 12 N</td><td>ThermoFisher</td><td>S25358</td><td>HCl</td></tr><tr><td>1-Methylimidazole</td><td>Sigma Aldrich</td><td>336092</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;prime;-ethylcarbodiimide hydrochloride</td><td>Sigma Aldrich</td><td>39391</td><td>EDC</td></tr><tr><td>Hydrogen peroxide solution H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>216763</td><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Trisodium citrate dihydrate</td><td>Sigma Aldrich</td><td>S1804</td><td>Sodium Citrate</td></tr><tr><td>miRCURY LNA miRNA ISH Buffer Set (FFPE)</td><td>Qiagen</td><td>339450</td><td>scramble miRNA/U6 snRNA</td></tr><tr><td>miRCURY LNA mmu-miR-182 detection probe</td><td>QIagen</td><td>YD00615701</td><td>5&#039;-DIG and 3&#039;-DIG labelled</td></tr><tr><td>Levamisol hydrochloride</td><td>Sigma Aldrich</td><td>31742</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9647</td><td>BSA</td></tr><tr><td>NBT/BCIP Tablets</td><td>Sigma Aldrich</td><td>11697471001</td><td>NBT-BCIP</td></tr><tr><td>Potassium Chloride</td><td>ThermoFisher</td><td>P217</td><td>KCl</td></tr><tr><td>DAPI solution (1mg/ml)</td><td>ThermoFisher</td><td>62248</td><td>DAPI</td></tr><tr><td>Glass coverslip</td><td>ThermoFisher</td><td>12-545E</td><td>Glass coverslip</td></tr><tr><td>Plastic coverslip</td><td>Grace Bio-Labs</td><td>HS40 22mmX40mmX0.25mm</td><td>RNA-ase free plastic coverslip</td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Sigma Aldrich</td><td>11093274910</td><td>DIG antibody</td></tr><tr><td>Hydrophobic barrier pen</td><td>Vector Laboratories</td><td>H-4000</td><td>Pap pen</td></tr><tr><td>Anti-Cardiac Troponin T antibody</td><td>Abcam</td><td>ab92546</td><td>cTnT antibody</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, Alexa Fluor 568</td><td>ThermoFisher</td><td>A-11011</td><td>anti-rabbit-568 antibody</td></tr><tr><td>Dako Fluorescence Mounting Medium</td><td>DAKO</td><td>S3023</td><td>mounting medium</td></tr><tr><td>Sheep serum</td><td>Sigma Aldrich</td><td>S3772</td><td></td></tr><tr><td>Goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td></td></tr><tr><td>Deionized Formamide</td><td>American Bioanalytical</td><td>AB00600</td><td></td></tr><tr><td>Hybridization Oven</td><td>ThermoFisher</td><td>UVP HB-1000 Hybridizer</td><td></td></tr></tbody></table>

tissue-specific mirna expression profiling in mouse heart sections using in situ hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

miRNA in situ hybridization was optimized on mouse heart sections using a scramble miRNA and U6-snRNA, which served as negative and positive controls respectively. Positive staining is indicated in blue, while the negative staining is indicated by the lack of color development (Figure 1A-1B). Cardiomyocyte specific expression of miRNA-182 was assessed in heart sections from control and PlGF overexpressing mice. The mice carrying the ...

Watch this Scientific Journal Video about Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization at JoVE.com

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

tissue-specific-mirna-expression-profiling-mouse-heart-sections-using

Research

JoVE Journal

Genetics

8.1K Views.  University College London. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het microRNA-veld, door het verstrekken van tools om de relatieve lcalisatie van zowel eiwit- als microRNA-moleculen te detecteren. Het belangrijkste voordeel van deze techniek is dat u zowel eiwit- als microRNA-moleculen detecteren uit dezelfde weefselsectie. Dit protocol begint met een rehydratie van de op de glijbaan gemonteerde weefsels.Verwarm de glijbanen op 60 graden Celsius gedurende 10 minuten in de hybridi...

Video: Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Developmental Biology

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Biochemistry

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Neuroscience

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Medicine

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Biology

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app.jove.com/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

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