MicroRNA Protocol for In-situ Hybridization
on Frozen Sections
Adapted by Dr. Aurora Esquela-Kerscher
Yale University, United States
I. Synthesis of DIG labeled LNA miRNA probe (miRCURY™ LNA Detection probe, ready-to-label)
A. Label the miRCURY™ LNA Detection probe:
1. Label 100 pmol of the LNA oligos for miRNA detection using the DIG Oligonucleotide Tailing Kit (Roche cat # 3-353-583)
miRCURY™ LNA Detection probes from Exiqon concentration: 25 μM stock = 25pmol/μL
miRCURY™ LNA Detection probe labeling reaction:
Add 100 pmol miRCURY™ LNA Detection probe and water to a volume of 9 μL
Add the following on ice:
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4.0 ul reaction buffer
4.0 ul CoCl2-solution
1.0 ul DIG-dUTP solution
1.0 ul dATP solution
1.0 ul 400 U Terminal transferase
Mix and centerfuge briefly
Incubate at 37°C for 30 minutes and place on ice.
2. Stop reaction by adding 5μL of 0.1M EDTA (pH 8.0)
3. Purify probe using a Sephadex G25 Column (Amersham Biosciences # 27-5325-01)
- Resuspend column by inversion
- Snap top off, then bottom off
- Spin 1000g, 1 minute in 1.5 ml tube and discard buffer
- Add probe to column
- Spin 1000g in fresh 1.5 ml tube, 4 minutes and retain labeled probe
Approximate probe concentration = 30ng/ul Avoid freeze thaws by aliquotting labeled probe into multiple tubes before storage at –20°C.
4. Check labeling efficiency and approximate concentration by dot blot:
- dilute labeled LNA oligo and Control DIG-dUTP/dATP tailed LNA oligo (2.5 pmol/μL - provided in Roche kit) in water at the following serial dilutions: 1:10 5:50 5:50 5:50 5:50
- Spot 1 μL of undiluted probe and 1 μL of each dilution on nitrocellulose.
- Air dry membrane and UV-crosslink both sides
- Equilibrate with 1xPBS for 3’
- Block with 1% BSA in 1xPBS for 30’ at RT
- Incubate in block solution with 3 μL of anti-DIG-AP conjugated antibody for 30’ at RT (Roche Anti-Digoxigenin-AP #1093274)
- Wash 2X with 1xPBS for 10’ each
- Add 1-STEP NBT/BCIP (Pierce #34042) developer for 15’-30’
- Stop reaction with TE
II. Preparing Frozen Sections for Hybridization
A. Tissue Preparation
a) Dissect tissue and put into 4% Paraformaldehyde/PBS O/N at 4°C.
b) 0.5M Sucrose/PBS (filter sterilized) O/N (O/N: overnight) 4°C. (put tissue directly into sucrose without washing – residual 4% Paraformaldehyde is good)
c) Freeze tissue in OCT in an Ethanol/Dry Ice slurry and store blocks at –8O°C until ready to use.
B. Cut Sections for Hybridization
Schaeren-Weimers and Gerfin-Moser, Histochemistry (1993) 100:431-440
a) Cut sections of 12-14 uM on cryostat and mount on Superfrost /Plus (Fisher #1255015) slides.
b) Air dry sections for at least 20 minutes but no longer than 3 hours. (Store at –20°C) *Some people claim best to cut tissue and hybridize on same day but slides are fine at –20°C (frost-free freezer) for months.
c) Fix sections in 4% Paraformaldehyde/PBS at 4°C for 10 minutes.
d) Wash 3X with PBS for 5 minutes each.
e) Acetylate for 10 minutes in:
2.33 ml triethanolamine
500 ul acetic anhydride
volume up to 200 mls in water
* mix and use solution immediately on sections.
f) Wash with 1xPBS, 3X for 5 minutes each.
III. Hybridizing DIG Probes to Slides
A. Prepare Hybridization Mixture
a) Prepare hybridization mixture by adding 1-2 μL of labeled probe per 200 μL of hybridization buffer. (~30ng/ul = original conc of DIG probe)
Hybridization Buffer
D.Wilkinson In-situ Hybridization: Practical Approach, 1992
50% deionised formamide
0.3M NaCl
20 mM Tris HCL, pH 8.0
5 mM EDTA
10mM NaPO4, pH 8.0
10% Dextran Sulfate
1X Denhardt’s
0.5 mg/ml yeast RNA
Store in aliquots at –80°C
b) Heat Hybridization mixture at 65°C for 5 minutes to linearize probe and chill on ice.
c) Add 50-100 ul of hybridization solution to slides. Spread solution over section gently with a piece of parafilm.
d) Cover sections with RNase-free plastic coverslips. (Hybrislip, Schleicher & Schuell, HS40 – 40x22mm; HS22 – 22x22mm via Thomas Scientific)
e) After "coverslipping", incubate slides O/N at a temperature that is 20-22°C below Tm (Tm: melting temperature) of the miRCURY™ LNA Detection probe in a chamber humidified* with:
50% formamide
1X SSC
(soaked Kimwipes)
*humidified chamber: small plastic slide box with lid (Micro Slide Box, VWR # 48444004) and soaked kimwipe placed at the bottom of box, taped closed around the edges and placed in incubator or hyb oven.
B. Washing Slides After Hybridization
(adapted from Heller et al. PNAS 95:11400-11405)
a) Remove coverslips in 5X SSC at RT. (15-20 minutes)
b) Wash slides 2X for 30 minutes each at a temperature that is 20-22°C below Tm of the miRCURY™ LNA Detection probe in:
50% formamide
0.1% Tween-20
1X SSC
c) Wash slides for 15 minutes in 0.2 X SSC at RT
d) Wash slides for 15 minutes in PBS at RT
C. Antibody Detection
(adapted from Heller et al. PNAS 95:11400-11405)
a) Incubate slides for 1 hr at RT in Blocking Solution:
0.5% blocking powder (Blocking Reagent, Roche # 1096176)
10% heat-inactivated goat (or sheep) serum (70°C for 30 minutes)
0.1% Tween-20
1X PBS
• Heat gently to get into solution
(but overheating results in "crashing" out of solution)
b) Incubate slides for 2-3 hours at RT in blocking solution preincubated for 1 hour with AP conjugated anti-DIG Fab fragment (1:1500 – Roche #1093274).
Can use a PAP Pen (Liquid Blocker Super PAP Pen) to draw a liquid-repellent barrier around section on slide to decrease amount of Antibody solution used per slide.
Place slides in tupperware container with damp (with distilled water) paper towels on the bottom to make a humidified chamber.
c) Washes:
2X 30 minutes in 0.1% Tween-20 in PBS
2X 20 minutes in PBS
d) Detection:
Incubate 250 μL of 1-STEP NBT/BCIP (Pierce #34042) or BM Purple AP Substrate (Roche #11442074001) together with 2mM Levamisole on slide for 1-4 days in the dark at RT.
* To perform color reaction, place slides upside-down with ~250μL of AP Substrate on top of a Styrofoam tray (can use inverted old 50ml Falcon tube rack) that is covered with two pieces of parafilm. The inverted styrofoam tray is placed into a tupperware container that has distilled water just covering the bottom (thus making a humidified container). Prevent the AP substrate from contacting air by placing a parafilm sheet over the surface of slides in order to avoid the formation of nonspecific precipitate due to oxidation of the substrate.
When desired intensity is reached, stop the reaction by transferring slides into Stop Solution (PBS pH 5.5, EDTA 1mM.)
Mount slides in a water-based medium (Glycerol Gelatin – heated approx 45°C or Aqua Mount, Lerner Labs #13800 - via Fisher #143905 - used at RT)
Troubleshooting:
a) In some situations using single 3’ DIG-labeled LNA oligos might decrease background compared to the use of the 3’-End DIG labeling kit (Roche cat # 3-353-575). Pre-labeled miRCURY™ LNA Detection probes can be bought from Exiqon.
b) Substituting 0.1% Triton X-100 for 0.1% Tween-20 in the washes might help to cut down the background. I have found that 0.1% Tween-20 work fine for my miRCURY™ LNA Detection probes.
c) If AP substrate detection results in a brown rather than purple/blue staining, it might be worth trying the following: stain O/N with 1-Step NBT/BCIP followed by O/N stain with BM AP substrate purple, wash and mount.
