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MicroRNA detection in FPPE Sections by in situ hybridization
Sponsored,vendor-submitted protocol    Sponsored by Exiqon A/S    Published in BioTechniques Protocol Guide 2007 (p.75) DOI: 10.2144/000112356

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:

  1. Xylene 3× 5min
  2. Ethanol 100% 2× 5min
  3. Ethanol 70% 5min
  4. Ethanol 50% 5min
  5. Ethanol 25% 5min
  6. DEPC 1× 1min

Sections are deproteinated:

  1. 2× 5 min phosphate buffered saline (PBS)
  2. 5 min Proteinase K at 10ug/ml at 37°C (add Proteinase K, 20mg/ml, to warm Proteinase K buffer 20 min before incubation)
  3. 30sec 0.2% glycine in PBS
  4. 2×30sec PBS
  5. Fix sections for 10 min in 4% paraformaldehyde
  6. Rinse slides 2× in PBS


  1. 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)

  1. Dilute probe to 20 nM in hybridization buffer
  2. Add 200 µl hybridization mix per slide
  3. Hybridize slides overnight covered with Nescofilm in a humidified chamber

Stringency Wash

  1. Rinse in 2× SCC at hybridization temperature
  2. Wash 3×30 min in 50% formamide, 2×SSC at hybridization temperature
  3. 5×5 min in PBST at RT

Immunological Detection

  1. 1 h block in blocking buffer consisting of 2% sheep serum, 2mg/ml BSA in Phosphate buffer saline tween-20 (PBST) at RT
  2. Overnight antibody incubation (1:2000 anti-DIG-AP Fab fragments in blocking buffer) in a humidified chamber at 4°C
  3. Wash 5–7 times 5 min in PBST
  4. Wash 3 times 5 min in AP buffer


  1. light sensitive color reaction (NBT/BCIP) 1h–48h (400ul/slide) in a humidified chamber
  2. wash slides 3×5 min in PBST
  3. mount in aqeous mounting medium (glycerol) or dehydrate and mount in Entellan.


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.

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