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protocol
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

Prehybridization

  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

COLOR REACTION (RT, darkness)

  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.

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.

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