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

The overall goal of this procedure is to perform a rapid in C two hybridization on paraform aldehyde prefixed brain tissue. This is accomplished by first dehydrating brain tissue sections and treating the sections with pretreatment one, two, and three reagents. The second step is to hybridize the sections with primary oligonucleotide probes and then second, third, and fourth probes.

Next, add fast red substrate to detect target RNA. The final step is to quantify the level of mRNA signals with Image J software. Ultimately, changes in RNA gene expression can be obtained by comparing the experimental and control groups.

The main advantage of this technique over existing methods is that it is relatively simple, inexpensive reproducible, and takes less than two days to complete. This work was developed by myself and John Jay Lenan from Ross University School of Veterinary Medicine and Jean Soza and RU Tao from the College of Medicine, Florida Atlantic University. In this procedure, her dehydration take four slides from microscope slide boxes stored in the negative 80 degree Celsius freezer.

Assign the sections to the tests of P-P-I-B-D-A-P-B and HT two A 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 pre-treatment 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 1000 milliliter beaker containing 450 milliliters of the pretreatment two reagent.

Next, boil the slides for a total of five minutes at 100 degree 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 three 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 P-P-I-B-D-A-P-B or HTR two A 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 one 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 two 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 three 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 four 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. Pipet 10 microliters of the amplification five 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. Pipet 10 microliters of the amplification six 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 cover slip.

In this step, drag and drop the image file into the main image J window. Go to the menu bar and select image. Next select type from the dropdown menu and select eight bit.

Next, select adjust then select threshold to open the dialogue box. Adjust the lower bar value in the dialogue box so that the unwanted background is removed. Go to the menu bar and select Analyze, then select analyze particles to open a second dialogue box.

Set the particle size to 10. Next, select show outlines and select display results and summarize boxes. Lastly, click okay to view particle data in the dialogue boxes.

For spreadsheet calculation, enter the data into the Excel sheet and average the number of particles in the saline group as a baseline. Then calculate the percentage level of each section and make a graph accordingly. Here are the light microscopic views of the hybridization signals indicated by the red dots.

Tissues were prefixed with paraform aldehyde in deeply anesthetized rats previously treated with saline and MDMA. The oligonucleotide probes used are D-A-P-B-P-P-I-B, and HT two A.This figure shows the effect on hypothalamic HT two A gene expression in rats previously treated with MDMA and a 20%reduction in HT two, a expression was observed. This technique makes it easier for researchers in the field of neuroscience to measure messenger R a's gene expression, using in situ ization on paraform, the height prefixed brain tissue.