Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

Summary

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT_2A receptor gene htr2a in rats.

Abstract

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT_2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT_2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Introduction

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants¹, while also occurring in situations of MDMA use for recreational purposes². Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood^3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment^5,6.

Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes⁷. Furthermore, the probes produce a low background signal without sacrificing specificity⁸. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.

Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain^5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT_2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

Protocol

Animal use procedures described below were approved by The Institutional Animal Care and Use Committee (IACUC) at Ross University School of Veterinary Medicine and Florida Atlantic University. Although sterile techniques and gloves are required, the RNase-free environment is not necessary while using this protocol.

1. Preparation

1. Tissue preparation and sectioning
   1. Assign rats randomly into three groups: fresh/saline (SAL; 0.9% NaCl), prefixed/SAL and prefixed/3,4-methylenedioxymethamphetamine (MDMA; see details in table 1).
   2. 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.
   3. 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).
   4. For the fresh/SAL group, quickly decapitate rats and remove the brain for the fresh brain test (see details in the reference⁵).
   5. 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.
   6. 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.
   7. 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 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; see the reference⁹).
   8. 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.
   9. Turn on the paraformaldehyde pump at the medium level and allow the ice-cold 4% paraformaldehyde to perfuse the animal for 30 min.
   10. 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 ¹⁰.
   11. Remove the brain with a spatula from the skull cavity and place the brain in a 50 ml centrifuge tube containing 25 ml of 4% paraformaldehyde. Keep the brain within the paraformaldehyde-containing tube at 4 ºC refrigerator for 24 hr.
   12. Move the brain into a 50 ml centrifuge tube containing 25 ml of 30% sucrose solution at 4 ºC for 3 days. Finally, store the brain within plastic bags at -80 °C till use.
   13. Mount the brain on a chuck with embedding media and freeze the embedding media containing brain in a cryostat at temperature of -26 ºC. Cut the fresh frozen brain into 20 µm sections and paraformaldehyde-prefixed brain into 10-µm sections. Transfer the section to a RT microscope slide by touching the slide to the section.
   14. Air-dry at RT for 4 hr. Store the section slides in a tight sealed bag at -80 °C till use.
2. Dehydration
   1. Take slides from the -80 °C freezer and assign sections into one of three tests: ht2a, ppiB (positive control) or dapB (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.
3. Pretreatment
   1. Pipette 20 µl 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).
   2. 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 ºC (heat-induced restoration of RNA structure¹¹).
   3. 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 Figure 2).
   4. Pipette 10 µl 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 ºC; gently shake the tray for 30 min. Wash the slides twice with ddH₂O for 2 min.

2. Hybridization and Amplification

1. Pipette 10 µl of htr2a, ppiB and dapB 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 ºC in the oven for 2 hr.
2. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl of the Amplification-1 reagent on each selected area, shake and incubate the slides at 40 ºC for 30 min.
3. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl of the Amplification-2 reagent on each selected area, shake and incubate the slides at 40 ºC for 15 min.
4. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl of the Amplification-3 reagent on each selected area, shake and incubate the slides at 40 ºC for 30 min.
5. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl of the Amplification-4 reagent on each selected area, shake and incubate the slides at 40 ºC for 15 min.
6. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl of the Amplification-5 reagent on each selected area, shake and incubate the slides at RT for 30 min.
7. Wash the slides twice with a washing buffer for 2 min. Pipette 10 µl 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.

3. Signal Detection

1. Pipette 10 µl 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.
2. Wash the slides twice with (ddH₂O) for 10 min. Dry the slides at 60 ºC in the oven for 15 min. Place the slides in xylene under the fume hood for 5 min. Pipette 20 µl of xylene-based mounting media on each selected area and cover with a coverslip.

4. Image Capture

1. Take digital microphotographs of the selected areas (e.g., hypothalamus, Figures 1 and 3) with a magnification of 4× and 20× objective lenses. Save the image file.

5. Image Particle Analysis

1. Drag and drop the image file into the main ImageJ window. Go to the menu bar and select ‘Image’, next select ‘Type’ from the drop-down menu, and select ‘8-bit’.
2. Select ‘Adjust’, then select ‘Threshold’ to open the dialog box. Adjust the lower bar value in the dialog box so that the unwanted background is removed. Go to the menu bar and select ‘Analyze’, then select ‘Analyze Particles’ to open a second dialog box.
3. Set particle size at 10, next select ‘Show outlines’ and select ‘Display results” and ‘Summarize’ boxes. Lastly, click ‘OK’ to view particle data in the dialog boxes.

6. Spreadsheet Calculation

1. Enter the data into the Excel sheet and average the numbers of particles in the SAL group as a baseline. Calculate the percentage level of each section and make a graph accordingly (Figure 4).

Results

Using the oligonucleotide RNA probes (<25 nt), hybridization can be detected as red dots in hypothalamic cells prepared from the paraformaldehyde-prefixed and fresh frozen brains. The htr2a mRNA molecules are present in some cells (indicated by solid arrows), but not others (open arrows). We observed that there is no difference between the paraformaldehyde-prefixed and the fresh frozen tissues (Figure 1). A successful hybridization can be evaluated first by the naked eye (Figure 2

Discussion

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution^5,12. Although quick preservation is critical for hybridization using a riboprobe¹³, little is known about experimental conditions...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RNAscope Negative Control Probe-DapB	Advanced cell diagnostic, INC	310098
RNAscope Pretreat 4	Advanced cell diagnostic, INC	320046
RNAscope 2.0 HD Reagent Kit - Red	Advanced cell diagnostic, INC	310036
RNAscope Probe - Rn-Ppib	Advanced cell diagnostic, INC	313921
Rat Htr2a	Advanced cell diagnostic, INC	300031
Cryostat	Leica	CM 1850
Horizontal shaker	VWR	88032-088
Hybridization oven	Thermo Fisher Scientific	222000
Superfrost Plus microscope slide	Fisher Scientific	12-550-15
Hydrophobic pen	Vector	H-4000
Microscope	Olympus	Provis AX70