Name: in situ hybridization using oligonucleotide probes to study rna expression in rat brain sections
Uploaded: 2025-03-31T15:00:40.000+00:00
Duration: 1 min 29 s
Description: Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

in situ hybridization using oligonucleotide probes to study rna expression in rat brain sections

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

In this procedure, for dehydration, take four slides for microscope slide boxes stored in the negative 80 degree Celsius freezer. Assign the sections to the tests of ppiB, dapB, and ht2a genes. Mark and label them with a ballpoint pen.
Next, submerge the slides in 100% alcohol at room temperature for five minutes. Then, dry the slides for five minutes in the fume hood. For pretreatment, pipette 20 microliters of the pretreatment one reagent to each section.
Place the slides on a rack rail in a moisture tray. Gently shake the moisture tray on a horizontal shaker at low speed for 10 minutes. Then, wash the slides twice with double distilled water for two minutes. Submerge the slides in a 1,000-milliliter beaker containing 450 milliliters of the pretreatment 2 reagent.
Next, boil the slides for a total of five minutes at 100 degrees Celsius to restore the RNA structure. After that, wash the slides twice with double distilled water for two minutes each time. Place the slides in 100% alcohol for five minutes.
Then dry the slides for five minutes in the fume hood. Use a hydrophobic pen to draw a circle around the selected area in the tissue section. Subsequently, pipette 10 microliters of the pretreatment reagent to each selected area to increase probe penetration, and cover the moisture tray with a lid.
Next, place the tray in the hybridization oven equipped with a horizontal shaker. Set the oven temperature to 40 degrees Celsius and gently shake the tray for 30 minutes. Afterward, wash the slides twice with double distilled water for two minutes each time.
In this step, pipette 10 microliters of ppiB, dapB, or htr2a probe on the selected areas. Then, cover the moisture tray with a lid to prevent evaporation of the reagent. Gently shake the slides on the horizontal shaker at low speed while incubating them at 40 degrees Celsius in the oven for two hours.
Next, wash the slides twice with washing buffer for two minutes each time. Pipette 10 microliters of the amplification 1 reagent on each selected area. Shake, and incubate the slides at 40 degrees Celsius for 30 minutes. Then, wash the slides twice with washing buffer for two minutes each time.
Pipette 10 microliters of the amplification 2 reagent on each selected area. Shake, and incubate the slides at 40 degrees Celsius for 15 minutes. After that, wash the slides twice with washing buffer for two minutes each time.
Pipette 10 microliters of the amplification 3 reagent on each selected area. Shake, and incubate the slides at 40 degrees Celsius for 30 minutes. Then wash the slides twice with washing buffer for two minutes.
Pipette 10 microliters of the amplification 4 reagent on each selected area. Shake, and incubate the slides at 40 degrees Celsius for 15 minutes. Next, wash the slides twice with washing buffer for two minutes.
Pipette 10 microliters of the amplification 5 reagent on each selected area. Shake and incubate the slides at room temperature for 30 minutes. Subsequently, wash the slides twice with washing buffer for two minutes.
Pipette 10 microliters of the amplification 6 reagent on each selected area. Shake, and incubate the slides at room temperature for 15 minutes. Then, wash the slides again twice with washing buffer for two minutes each time.
Now, pipette 10 microliters of the red reagent to each selected area. Place the slides in the moisture tray on the horizontal shaker at room temperature, and shake gently at low speed for 10 minutes. Then, wash the slides twice with double distilled water for 10 minutes each time.
Dry the slides at 60 degrees Celsius in the oven for 15 minutes. After that, place the slides in xylene in the fume hood for five minutes. Next, pipette 20 microliters of xylene-based mounting media on each selected area and cover with a coverslip.

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In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation
<ol>
	<li>Tissue preparation and sectioning
	<ol>
		<li>Assign rats randomly into three groups: fresh/saline (SAL; 0.9% NaCl), prefixed/SAL and prefixed/3,4-methylenedioxymethamphetamine (MDMA; see details in&#160;table 1).</li>
		<li>Administer three doses of 1 ml/kg SAL and 10 mg/kg MDMA intraperitoneally, respectively, to the SAL and MDMA groups at 2 hr interval. Allow animals to survive the treatment for 6 days.</li>
		<li>On day 7, administer 100 mg/kg ketamine in combination of 5 mg/kg xylazine intraperitoneally to the rats for a deep anesthesia (i.e., loss of corneal blink and tail pinch reflexes).</li>
		<li>For the fresh/SAL group, quickly decapitate rats and remove the brain for the fresh brain test.</li>
		<li>For the prefixed/SAL and prefixed/MDMA groups, make a cut along the sternum about 10 -12 cm and then expose the end of the sternum.</li>
		<li>Hold the end of the sternum with a hemostat and cut the diaphragm laterally on both sides with sharp scissors and then cut upward across the ribs and parallel to the lungs.</li>
		<li>With one hand, hold the ventral tip of the heart with small forceps. With the other hand, pierce the left ventricle with a 18 G&#160;syringe needle (note the adapter side of the needle is connected with a Y-shaped silicon hose to ice-cold SAL and paraformaldehyde containers and peristaltic pumps).</li>
		<li>Turn on the SAL pump at the medium level of the flow rate (~5 ml/min) and puncture the right atrium with the forceps to allow the escape of return circulation. Maintain SAL perfusion for 30 min.</li>
		<li>Turn on the paraformaldehyde pump at the medium level and allow the ice-cold 4% paraformaldehyde to perfuse the animal for 30 min.</li>
		<li>Remove perfusion instrument and decapitate the animal. Make a posteroanterior cut through the midline of the skull, and then flap the skull to expose the skull cavity.</li>
		<li>Remove the brain with a spatula from the skull cavity and place the brain in a 50&#160;ml centrifuge tube containing 25 ml of 4% paraformaldehyde. Keep the brain within the paraformaldehyde-containing tube at 4 &#186;C refrigerator for 24 hr.</li>
		<li>Move the brain into a 50&#160;ml centrifuge tube containing 25 ml of 30% sucrose solution at 4 &#186;C for 3 days. Finally, store the brain within plastic bags at -80 &#176;C till use.</li>
		<li>Mount the brain on a chuck with embedding media and freeze the embedding media containing brain in a cryostat at temperature of -26 &#186;C. Cut the fresh frozen brain into 20&#160;&#181;m sections and paraformaldehyde-prefixed brain into 10-&#181;m sections. Transfer the section to a room temperature (RT) microscope slide by touching the slide to the section.</li>
		<li>Air-dry at RT for 4 hr. Store the section slides in a tight sealed bag at -80 &#176;C till use.</li>
	</ol>
	</li>
	<li>Dehydration
	<ol>
		<li>Take slides from the -80 &#176;C freezer and assign sections into one of three tests:&#160;serotonin 5-HT2A&#160;receptor gene (ht2a),&#160;ppiB&#160;(peptidylprolyl isomerase B gene, positive control) or&#160;dapB&#160;(bacterial dihydrodipicolinate reductase gene, negative control); mark and label sections with a ball pen (do not use a pencil). Submerge slides in 100% alcohol at RT for 5 min. Dry the slides for 5 min in a fume hood.</li>
	</ol>
	</li>
	<li>Pretreatment
	<ol>
		<li>Pipette 20 &#181;l of the Pretreatment-1 reagent (inactivation of alkaline phosphatase) to the sections on slides. Place the slides on a rack rail in a moisture tray (note that the tray is covered with a lid to maintain a high humidity for preventing evaporation from the reaction solution).</li>
		<li>Gently shake the moisture tray on a horizontal shaker at a low speed for 10 min. Wash the slides twice with double-distilled water (ddH2O) for 2 min. Submerge the slide in a 200 ml beaker containing 150 ml of the Pretreatment-2 reagent (breaking of the cross-linkages induced by paraformaldehyde fixation). Boil the slides for a total of 5 min at 100 &#186;C (heat-induced restoration of RNA structure).</li>
		<li>Wash the slides twice with ddH2O for 2 min. Place the slides in 100% alcohol for 5 min. Dry the slides for 5 min in the fume hood. Use a hydrophobic pen to draw a circle around the selected area in the tissue section (e.g., non-cortical structures, including thalamus and hypothalamus as outlined in&#160;Figure 1).</li>
		<li>Pipette 10 &#181;l of the Pretreatment-3 reagent (increases in probe penetration) to each selected area and cover the moisture tray with the lid. Place the tray in the hybridization oven equipped with the horizontal shaker and set the oven temperature at 40 &#186;C; gently shake the tray for 30 min. Wash the slides twice with ddH2O for 2 min.</li>
	</ol>
	</li>
</ol>
2. Hybridization and Amplification
<ol>
	<li>Pipette 10 &#181;l of&#160;htr2a,&#160;ppiB&#160;and&#160;dapB&#160;probe reagents, respectively, on the selected areas. Cover the moisture tray with the lid to prevent evaporation of the reagent. Gently shake on the horizontal shaker set at a low speed. Incubate the slides at 40 &#186;C in the oven for 2 hr.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-1 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-2 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 15 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-3 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-4 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 15 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-5 reagent on each selected area, shake and incubate the slides at RT for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-6 reagent on each selected area, shake and incubate the slides at RT for 15 min. Wash the slides twice with a washing buffer for 2 min.</li>
</ol>
3. Signal Detection
<ol>
	<li>Pipette 10 &#181;l of the Red reagent to each selected area (Note that the reagent is a mixture of Red-B and Red-A at a 1:60 ratio and should be used immediately). Place the slide in the moisture tray on the horizontal shaker at RT and shake gently at a low speed for 10 min.</li>
	<li>Wash the slides twice with (ddH2O) for 10 min. Dry the slides at 60 &#186;C in the oven for 15 min. Place the slides in xylene under the fume hood for 5 min. Pipette 20 &#181;l of xylene-based mounting media on each selected area and cover with a coverslip.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RNAscope Negative Control Probe-DapB</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310098</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Pretreat 4</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>320046</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - Red</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310036</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Probe - Rn-Ppib</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>313921</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Htr2a</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>300031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM 1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>VWR</td>
			<td>88032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>222000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Provis AX70</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization using oligonucleotide probes to study rna expression in rat brain sections

Source: Shokry, I. M., et al. <a href="https://app.jove.com/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Table 1.&#160;Experimental design
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Fresh</td>
			<td colspan="2">Paraformaldehyde-prefixeda</td>
		</tr>
		<tr>
			<td></td>
			<td>SALb</td>
			<td>SALb</td>
			<td>MDMAc</td>
		</tr>
		<tr>
			<td>Target probe (htr2a)</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>Positive probe (<em...

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

Take fixed brain sections from a rat with serotonin syndrome, which leads to altered serotonin receptor RNA synthesis in the neurons.
Dehydrate the tissue using alcohol. Apply a pretreatment solution to deactivate endogenous hydrolyzing enzymes, preventing their interaction with detection reagents.
Treat with a boiling buffer to break fixation-induced crosslinks, exposing the target RNAs.
Dehydrate the tissue, draw a hydrophobic barrier, and incubate it with a solution to permeabilize cellular membranes.
Add oligonucleotide probes and incubate, allowing the probes to hybridize with the target RNAs.
Wash to remove unbound probes, then incubate with amplification reagents containing preamplifier, amplifier, and enzyme-conjugated label probes that bind to the oligonucleotide probes.
Incubate with chromogenic substrates that get hydrolyzed by the enzyme-conjugated probes, producing visible color.
Treat with xylene to increase tissue transparency, add a mounting medium, and apply a coverslip.
Under a microscope, colored spots in the tissue help quantify target RNA expression.

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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

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In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

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In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections