This work was supported by US National Institutes of Health grants HL082798 and HL111580.

1. Kidney Tissue Sections
<ol>
	<li>Formalin-fixed (10%) paraffin-embedded kidney sections are cut onto microscope slides at 5 mm thickness using a Microm HM 355 S. The slides can be stored for several months before analysis.</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare 1 L of 1x PBS in ddH2O in an autoclavable screw top bottle. Fill another bottle with 1 L of ddH2O. Add 1 ml of DEPC to each bottle, cover and shake vigorously. Let the solutions sit overnight at room temperature to destroy RNAses and then autoclave. Add 400 ml Tween-20 to 1 L of 1x PBS to make PBS-T.</li>
	<li>Prepare proteinase K buffer (50 mMTris-HCl, pH 8.0 and 1 mM CaCl2 in DEPC treated water).</li>
	<li>Prepare a solution of 0.2% glycine in PBS-T.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>Prepare a large volume of hybridization buffer (50-100 ml) composed of 50% Formamide, 5x SSC, 0.1% Tween, 9.2 mM citric acid for adjustment to pH 6, 50 &#181;g/ml heparin, and 500 &#181;g/ml yeast RNA in DEPC treated ddH2O. Divide into 1 ml aliquots and store at -80 &#186;C.</li>
	<li>Remove the paraffin from the tissue by washing 3x in fresh xylene for 5 min each.</li>
	<li>Rehydrate the tissue sections by 5 min incubations in decreasing concentrations of ethanol (100%, 75%, 50%, and 25%) in ddH2O.</li>
	<li>Treat the slides with enough DEPC to completely cover the tissue sections (approximately 0.4-0.5 ml) for 1 min. Make sure tissues do not dry out.</li>
	<li>Rinse the slides in DEPC treated PBS-T twice for 5 min, collecting the DEPC containing waste for proper disposal. All reagents should be DEPC treated to eliminate RNAses from this point on.</li>
	<li>Prewarm Proteinase K buffer and add proteinase K to a final concentration of 10 &#956;g/ml. Cover tissue sections with proteinase K solution and incubate at 37 &#186;C for 5 min.</li>
	<li>Quickly remove the proteinase K solution and add 0.2% glycine in PBS-T for 30 sec.</li>
	<li>Rinse slides in DEPC treated PBS-T twice for 30 sec each.</li>
	<li>Fix sections in freshly made 4% paraformaldehyde for 10 min.</li>
</ol>
4. Hybridization
<ol>
	<li>Add 50 &#181;l of hybridization buffer (50% Formamide, 5x SSC, 0.1% Tween, 9.2 mM citric acid for adjustment to pH 6, 50 &#181;g/ml heparin, 500 &#181;g/ml yeast RNA) per tissue section and cover with RNAse free HybriSlips cut just larger than the tissue sections.</li>
	<li>Prehybridize sections in humidity controlled hybridization oven for 2 hr at hybridization temperature determined for the 3&#8217; digoxigenin labeled probe (usually DNA Tm of the probe -21 &#176;C, ( i.e.&#160;54 &#176;C for miR-382, probe DNA Tm= 75 &#176;C), using tissue lab wipes soaked in 50% formamide/50% 5x SSC to keep the chamber humidified.</li>
	<li>Dilute the miRNA probe, positive control (U6, small nuclear RNA) and negative control (scrambled sequence) to 40 nM in hybridization buffer (75 &#181;l of buffer is needed per section).</li>
	<li>Heat the probe in hybridization buffer at 65&#160;&#186;C for 5 min.</li>
	<li>Remove slides from hybridization oven and remove coverslips and excess hybridization buffer from prehybridization.</li>
	<li>Add 75 &#181;l of probe containing buffer per tissue section, directly to tissue. Cover tissue with HybriSlips just large enough to cover the tissue sections.</li>
	<li>Keeping slide holder level, return to hybridization oven for overnight incubation at temperature from step 4.2 (approximately 16 hr).</li>
</ol>
5. Stringency Wash
<ol>
	<li>Add 200 &#181;l 2x SSC, heated to the hybridization temperature, just under one edge of the coverslips to help release the coverslip from the tissue. Remove the coverslip by tilting the slide.</li>
	<li>Wash the slides in 200 &#181;l of 50% formamide, 50% 2x SSC at hybridization temperature 3x for 30 min each.</li>
	<li>Rinse the slides 5x in PBS-T at room temperature for 5 min each on orbital shaker on low speed.</li>
</ol>
6. Detection
<ol>
	<li>Incubate slide in blocking buffer (2% goat or horse serum, 2 mg/ml BSA in PBS-T) for 1 hr at room temperature on orbital shaker on low speed.</li>
	<li>Dilute anti-DIG-AP Fab fragments in blocking buffer at 1:100. Add 100 &#181;l per tissue section and cover with HybriSlips cut just large enough to cover the section.</li>
	<li>Incubate antibody in a humidified chamber at 4 &#176;C overnight (approximately 16 hr).</li>
	<li>Wash the slides in PBS-T 7x, for 5 min each on orbital shaker at low speed.</li>
	<li>Wash the slides in AP buffer (100 mM TrisHCl pH 9.5, 50 mM MgCl2,100mM NaCl, 0.1% Tween 20) 3x, for 5 min each.</li>
	<li>Add NBT/BCIP solution to AP buffer (200 &#181;l/10 ml buffer)</li>
	<li>Add 400-500 ml of diluted NBT/BCIP solution to each slide to completely cover all tissue sections. Develop in a dark, level, humidified chamber for 4-5 hr.</li>
</ol>
7. Mounting Slides
<ol>
	<li>Dehydrate sections in increasing concentrations of ethanol (25%, 50%, 75%, 100%) for 5 min each.</li>
	<li>Incubate in xylene 5x in order to clear sections.</li>
	<li>Mount slides in Permount mounting medium and dry overnight.</li>
</ol>

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There is nothing to disclose.

The goal of this article was to describe a protocol for miRNA in situ hybridization that works well in formalin fixed kidney tissues. While working out this protocol several important sources of staining artifact have been identified. Careful attention to these points can help avoid staining artifact and increase the likelihood of a successful ISH run.
One of the most avoidable causes of staining artifact can occur when tissue sections become dried out during long incubations. When th...

MicroRNA are small (approximately 22 nucleotides long) noncoding RNAs that are endogenously produced. They generally function to suppress protein expression through translational repression or mRNA degradation. miRNAs bind to mRNA targets with incomplete complementarity, making it possible for a single miRNA to suppress multiple targets.
Understanding which cell types and structures express miRNAs is an important part of understanding the mechanisms through which alterations in miRNA expression influence cell and tissue phenotypes. While methods such as miRNA sequencing, qPCR and Northern blotting can be used for detection of miRNAs in whole tissues, this approach does not allow one to determine which specific cell type they came from within a given tissue. Dissection of cellular and structural components prior to analysis using these methods can be very difficult and the conditions needed to achieve adequate isolations can lead to alterations in gene expression or degradation of RNAs. microRNA in situ hybridization is a method used to visualize microRNA location and expression levels in tissues. This technique is especially valuable in tissues composed of heterogeneous structures, such as the kidney.
MicroRNAs have been shown to play an regulatory role in kidney development2,3 and physiology4. microRNA expression alterations have also been shown to be involved with renal pathology such as fibrosis5-10, diabetic nephropathy7, renal carcinoma11,12, and acute kidney injury13. In our research, we have found that optimizing microRNA in situ hybridization for kidney tissues was valuable for determining the exact structural locations of miRNA expression in both health and disease14. Determination of the tubular and cellular expression of different microRNAs is important because their regulation of targets may be dependent on cellular functions. In diseased states it is also important to determine how alterations in miRNA expression may be impacting function.
The goal of the method described here was to build upon existing ISH methodologies developed by Kloosterman et al.15, other investigators16,17, and those suggested by Exiqon1 and optimize the method for formalin fixed kidney tissues. We have successfully used this method to identify distinct regional differences in renal microRNA miR-382 expression with unilateral ureteral obstruction18. This approach can be used with other tissues as well, with additional optimization.

<table border="0" cellpadding="0" cellspacing="0" width="481">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
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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.

The areas of a tissue section that become dried out during the hybridizations, incubations or wash steps generally end up staining more darkly during the NBT/BCIP development. Figure 1 shows a portion of a kidney section in which the HybriSlip slipped off of the edge of the tissue, allowing it to become partially dehydrated. Despite rehydration and coverage in the remaining steps, the signal in the dehydrated portion is artificially high.
The importance of controlling the time...

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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.

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 ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

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JoVE Journal

Biology

14.8K Views.  Medical College of Wisconsin. This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.