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

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

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.
   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).
   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.
   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 room temperature (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: serotonin 5-HT_2A receptor gene (ht2a), ppiB (peptidylprolyl isomerase B gene, positive control) or dapB (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.
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 structure).
   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 1).
   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.

Results

Table 1. Experimental design

	Fresh	Paraformaldehyde-prefixeda
	SALb	SALb	MDMAc
Target probe (htr2a)	√	√	√
Positive probe (

Materials

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