This method can help answer key questions in the microRNA field, by providing tools to detect the relative lcalization of both protein and microRNA molecules. The main advantage of this technique is that you can detect both protein and microRNA molecules from the same tissue section. This protocol begins with a rehydration of the slide-mounted tissues.
Warm the slides at 60 degrees Celsius for 10 minutes in the hybridization oven. The paraffin should be visibly melting. Then, incubate the slides in a glass Coplin jar containing commercially-available clearing agent for 10 minutes.
Do this twice. Next, transfer the slides from wash to wash to rehydrate the tissues. Once hydrated, incubate the tissues in proteinase K working solution at 37 degrees Celsius for 15 minutes.
To remove the proteinase K, wash the slides in 1x PBS two times. Now, fix the tissues. First, make reservoirs on the slides using a hydrophobic pen to draw a water-repellent circle around the preparations.
Next, apply 500 microliters of 4%PFA solution within the barrier, and incubate the slides in a fume hood. To remove the fixative, wash the slides in a Coplin jar containing PBS for five minutes two times at room temperature. Next, wash the slides in 0.13 molar 1-Methylimidazole for 10 minutes.
Do this in the fume hood at room temperature, and perform this wash twice. Add the EDC fixative, and incubate at room temperature in a fume hood for one hour. Rinse off the slides with 50-millimolar Tris.
To prepare for the hybridization, first build a humidified chamber. Load two pieces of filter or tissue paper into the bottom of a box, and wet the paper with a one-to-one mixture of formamide and 1x SSC. Next, apply 200 microliters of pre-warmed hybridization solution to each slide, and incubate the humidified chamber of slides for one to three hours at 50 degrees Celsius.
After the incubation, replace the hybridization solution with 250 microliters of probe solution, and cover the slides with an RNase-free cover slip. Try to avoid trapping air bubbles under the cover slip. Now, carefully seal the chamber with parafilm, and incubate the slides overnight at approximately 30 degrees Celsius below the RNA melting temperature of the probe.
Optimization may be required. In addition to setting the right incubation temperature, different microRNAs are expressed at different levels, so optimization regarding the probe concentration may also be required to get the optimal signal. Following the hybridization, perform the stringency washes.
First, was in 2x SSC at 50 degrees Celsius for five minutes, or until the cover slips loosen. Then, perform two washes in 2x SSC at room temperature, each for five minutes. Then, continuing at room temperature, wash the slides for five minutes in 0.2x SSC, followed by five minutes in PBST, and finally, five minutes in 1x PBS.
At this point, the RNA-RNA hybrids are stable, and RNA-stringent conditions are no longer needed. For DIG antibody detection, first, touch up the hydrophobic barriers, and apply 500 microliters of blocking solution for thirty minutes. Incubate the slides at room temperature in a humidified chamber.
Next, remove the blocking solution and replace it with 500 microliters of DIG antibody solution. Incubate the slides at room temperature in a humidified chamber for an hour. Then, using Coplin jars, wash the slides three times in PBST for five minutes per wash.
Next, wash the slides in pre-staining solution for five minutes twice. Then, transfer the slides to a polypropylene slide mailer jar, and add 20 milliliters of AP substrate solution. Now, incubate the slides at 37 degrees Celsius for six to 24 hours, protected from the light.
When first using AP substrate, it is important to follow the reaction. On two-hour intervals, check the color development on the slides using a microscope, until an optimal incubation time has been determined. To remove the excess AP substrate, wash the slides twice in KTBT solution for five minutes per wash, followed by one wash in PBS for five minutes.
To detect the cTNT antibody, first, block the tissues for an hour. Then, apply 200 microliters of cTNT antibody solution, and incubate the slides overnight at 4 degrees Celsius in a humidified chamber. The next day, wash the slides in a Coplin jar containing PBS for five minutes.
Do this three times. Then, apply 250 microliters of anti-rabbit 568 antibody solution, and incubate the slides for an hour at room temperature in a humidified chamber. To wash out the excess secondary antibody, use three five-minute washes in PBS.
Then, if desired, apply 250 microliters of DAPI working solution, and incubate the slides for one minute, protected from light. To remove the excess DAPI stain, use two five-minute PBS washes, and after the washes, mount the slides. MicroRNA in-situ hybridization was optimized on mouse heart sections using a scrambled microRNA for negative control, and a U6 snRNA as a positive control.
Cardiomyocyte-specific expression of microRNA-182 was assessed in heart sections from control and PlGF-overexpressing mice. The mice carried a PlGF transgene under an alpha-MHC promoter, and developed cardiac hypertrophy, secondary to increased angiogenesis, within six weeks of transgene activation. The staining protocol revealed increased expression of microRNA-182 in the hearts of PlGF mice.
Next, to determine which cell types expressed microRNA-182, the same sections were immunostained for cTNT. DAPI staining was also used. In both control and PlGF mouse heart sections, microRNA-182 was found in the nuclear compartment of cTNT-positive cardiomyocyte cells.
After watching this video, you should have a good understanding of how to perform microRNAse hybridization, followed by protein immunostaining. While attempting this procedure, it is very important to remember to keep RNAse-free conditions throughout day one, during all steps leading up to prop hybridization. Following this procedure, other methods like Western blot in a real-time PCR can be performed to answer questions like, what are the relative expression levels of this particular microRNA in different tissues, or this particular protein in the same tissue section.
Don't forget that working with fixatives such as PFA and ADC can be extremely hazardous. Precautions, include wearing gloves and lab coats, as well as using fume hoods, should always be taken when performing this procedure.
