This article describes a detailed protocol for analyzing RNA expression in rat brain sections affected by serotonin syndrome. The method involves several steps including dehydration, pretreatment, hybridization, and amplification to visualize target RNAs.
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.
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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