Acknowledgement for the development of these protocols: Dr. Erno Wienholds and Dr. Wigard Kloosterman of the Hubrecht Laboratory, University of Utrecht.
Note: the following protocols have been developed using digoxygenin (DIG)-labeled miRCURY™ LNA in situ hybridization probes. However, the protocols are readily adaptable for miRCURY™ LNA in situ hybridization probes labeled with alternative detection moieties, such as fluorescent dyes, biotin, radioactive nucleotides etc.
miRCURY™ LNA detection probes pre-designed for all miRNAs are available pre-labeled with DIG, fluorescein and biotin. The probes are also available for enzymatic labeling with detection moieties in a ‘ready to label’ format.
Deparaffinize the sections:
- Xylene 3× 5min
- Ethanol 100% 2× 5min
- Ethanol 70% 5min
- Ethanol 50% 5min
- Ethanol 25% 5min
- DEPC 1× 1min
Sections are deproteinated:
- 2× 5 min phosphate buffered saline (PBS)
- 5 min Proteinase K at 10ug/ml at 37°C (add Proteinase K, 20mg/ml, to warm Proteinase K buffer 20 min before incubation)
- 30sec 0.2% glycine in PBS
- 2×30sec PBS
- Fix sections for 10 min in 4% paraformaldehyde
- Rinse slides 2× in PBS
Prehybridization
- 2 h in hybridization buffer (50% Formamide, 5×SSC, 0.1% Tween, 9.2mM citric acid for adjustment to pH 6, 50ug/ml heparin, 500ug/ml yeast RNA) in a humidified chamber (50% formamide, 5× SSC). Use DAKO Pen.
Hybridization (hybridization temperature = Tm probe −21°C)
- Dilute probe to 20 nM in hybridization buffer
- Add 200 µl hybridization mix per slide
- Hybridize slides overnight covered with Nescofilm in a humidified chamber
Stringency Wash
- Rinse in 2× SCC at hybridization temperature
- Wash 3×30 min in 50% formamide, 2×SSC at hybridization temperature
- 5×5 min in PBST at RT
Immunological Detection
- 1 h block in blocking buffer consisting of 2% sheep serum, 2mg/ml BSA in Phosphate buffer saline tween-20 (PBST) at RT
- Overnight antibody incubation (1:2000 anti-DIG-AP Fab fragments in blocking buffer) in a humidified chamber at 4°C
- Wash 5–7 times 5 min in PBST
- Wash 3 times 5 min in AP buffer
COLOR REACTION (RT, darkness)
- light sensitive color reaction (NBT/BCIP) 1h–48h (400ul/slide) in a humidified chamber
- wash slides 3×5 min in PBST
- mount in aqeous mounting medium (glycerol) or dehydrate and mount in Entellan.
Buffers
AP buffer (100 mM Tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20),
100ml Tris (100mM) 12.1g/l
20ml 5M NaCl (100mM) 5.84g/l
5ml 1M MgCl2 (5mM)
700ml sterile H2O, pH 9.5 and fill up to 1l
Color Solution (Light sensitive)
45ul 75mg/ml NBT (in 70% dimethylformamide)
35ul 50mg/ml BCIP-phosphate (in 100% dimethylformamide)
2.4mg Levamisole in 10 ml AP buffer.
