Visualizing miRNAs in adult skeletal muscle by hybridization in situ

1. Probe preparation
Use an in vitro transcription reaction optimized for making high specific activity miRNA sense or antisense riboprobe.

1). Assemble the following master mix per reaction (20 ml) in the “MM” tube for generation of miRNA sense and antisense RNA probes: 2.0ml 10X Transcription Buffer; 1.0ml 10mM ATP; 1.0ml 10mM CTP; 1.0ml 10mM GTP; 0.5ml 10mM UTP; 0.5ml 10mM digoxigenin-12-UTP; 2.5 ml (0.5mg/ml) DNA Template; 2.0ml T3/T7 RNA Enzyme Mix; 1.0ml SuperaseIn; 4.5ml Nuclease-free water
2). Incubate the reaction at 37^oC for 90 min. Add 1ml DNAse I to tube and incubate at 37^oC for 15-30 min to remove DNA template.
3). Add carrier tRNA, 2 ml 5.0M Ammonium Acetate and 3 volume of 100% ethanol. Mix well and put the tube on ice for 20 min.
4). Spin for 30 min at maximum speed in a 4^oC microfuge.
5). Discard the supernatant, and wash the pellet once with 70% ethanol.
6). Spin for 5 min at maximum speed in a 4^oC microfuge and discard supernatant.
7). Air-dry pellet for 5 min and dissolve pellet in 20µl of DEPC-treated water.
8). Check concentration of the sample on a spectrophotometer and dilute to 0.1-0.2µg/µl. Run 2-4µl of RNA probes on a 1.5% agarose gel to check the size and integrity of RNA probes.

2. Tissue preparation
Regeneration in mouse tibialis anterior (TA) muscle is induced by injection of 10 µl cardiotoxin. In situ hybridization with cRNA probes and immunocytochemistry were performed on serial cryosections of mouse TA. Sections could be stored at -80^oC for up to 2 months without significant variation in reactivity with cRNA probes and antibodies.

Fixation
1. Take the slides from –80^oC.
2. Fix the section in 4 % paraformaldehyde (in PBS) for 20 min, in a sterile jar, and in the hood.
3. In a new jar, wash the sections in PBS for 2 × 10 min.

Deproteinization with Proteinase K:
This step is necessary to make the tissue accessible for the probe. Proteinase K acts satisfactorily on cryo-sections yielding increased hybridization efficiency and retaining acceptable tissue morphology.

4. Prepare X ml of Proteinase K buffer and store it on ice, just prior to use add 1 µl/ml of proteinase K (20 mg/ml).
5. Take the slides out of the PBS-solution (one at the time), and apply appx. 200 µl Proteinase K solution on each slides (on the sections), and incubate each slide for 5-7.5 min. at room temp.
Important: at no point should the sections be left to dry out.
6. Post-fix the section in 4 % paraformaldehyde (in PBS) for 20 min, in a sterile jar, and in the hood.
7. Wash 2×5 min. in PBS (time not critical). Wash rapidly in DEPC-treated dH₂O.

Acetylation of the tissue
This step in useful to decrease background binding of the negatively charged radioactive probe by acetylation of the positively charged amino-groups in the proteins of the tissue.

6. Prepare 250 ml 0.1M TEA (3.35 ml TEA from a 7.5M stock solution).
7. Place the slides in a slide-rack and immerse them in TEA in a large staining jar with a magnet stirrer.
8. While stirring add acetic anhydride to a concentration of 0.25 % (625 µl) and incubate 10 min. at room temp.
9. Wash the slides in PBS for 5 min.
10. Wash the slides in 0.85 % NaCl for 5 min. (time not critical).

Dehydration
11. In a new jar dehydrate the slides in 30%, 50%, 70%, 85%, 95% and 100 EtOH (2 min. in each solution.
12. Let the slides dry, in a horizontal position for at least 1 hour.

3. Hybridization step
1). Pre-hybridization
Add pre-hybridization buffer on section and incubation is done at 30-40 for 2-4 hours.
2). Add probe into 1ml of Hybridization buffer and mix well by gently inverting tube. The final concentration of the probe is about 300-500ng/ml.
3). Aspire pre-hybridization buffer and add mixed hybridization solution on slides (100 µl for each slide), use parafilm to cover tissues and hybridize at 37-40^oC overnight in a moist chamber. Low hybridization must be used. If hybridization temperature is too high, there will be no hybridization.

4. Post-Hybridization Washes (Step 1-6 should be performed on shaker)
1. After hybridization, rinse slides with pre-warmed (37-40^oC) 4XSSC solution and remove parafilm.
2. Wash slides in 2XSSC at 37-40^oC for 20 min (in water bath with shaker).
3. Treat the slides with 400unit/ml (10 µg/ml) RNase A at 37^oC for 30 min.
4. Wash slides in 1XSSC at 40^oC for 10-15 min.
5. Wash slides twice times with 1XPBST for 5 min at room temperature.

5. Signal Detection (Steps should be performed on a shaker)
1. Incubate with alkaline phosphatase-coupled anti-digoxigenin antibody (alternative, FITC-coupled antibody, or another method of detection). Dilute the antibody 1:4000 in 1X PBST. Add 100µl per slide of the mixed antibody solution to the sample. Incubate at room temperature for 4 hours. If fluorescence is chosen as the detection method, protect the slides from light. Use Vecta Shield (BioValley) to mount the cover slips, and analyze by fluoresecent microscopy.

If alkaline phosphatase was chosen, follow the protocol:
2. Wash slides with 1XPBST in a staining jar 4 X30 min or overnight.
3. Wash slides with Developing Buffer for 20 min.
4. Develop Color. Add 4.5 ml of NBT, 3.6 ml of BCIP and 1 ml of levamisole in 1 ml of Developing Buffer and well mix the solution. Use 200µl of developing solution to cover tissue sections.
5. Add Developing solution on slides and incubate slides flat and allow precipitate to form. Observe signal detection periodically under a microscope. Stop reaction until good staining quality.
6. Add Developing solution on slides and incubate slides flat and allow precipitate to form. Observe signal detection periodically under a microscope. Stop reaction at good staining quality.

6. Post-Detection Washes
1. Wash with TE buffer for 5 min at RT to stop reaction. Wash the slides in 1XPBS for 20 min at RT.
2. Rinse with ddH₂O.
3. Mount slides with water based mounting medium, and view the hybridization signals under a light microscope.