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Non-radioactive in situ hybridization on paraffin sections using DIG-labeled miRCURY™ detection probes

 
Method prepared by Dr. Wigard Kloosterman, the Plasterk Group, Hubrecht Laboratory, Utrecht, The Netherlands
 
Deparaffinize the sections:
  1. xylene 3x 5min
  2. ethanol 100% 2x 5min
  3. ethanol 70% 5min
  4. ethanol 50% 5min
  5. ethanol 25% 5min
  6. 6. DEPC 1x 1min

Sections are deproteinated:

  1. 2x 5min PBS
  2. 5min Prot.K at 10ug/ml at 37oC (add Prot.K 20mg/ml to warm Prot.K buffer 20 min before incubation)
  3. 30sec 0.2% Glycine in PBS
  4. 2x30sec PBS
  5. Fix sections for 10 min in 4%PFA
  6. 6. Rinse slides 2x in PBS

Prehybridization

  1. 2h-in hybridization buffer (50%Formamide, 5xSSC, 0.1%Tween, 9.2mM citric acid for adjustment to pH6, 50ug/ml heparin, 500ug/ml yeast RNA) in a humidified chamber (50% formamide, 5xSSC). Use DAKO Pen.
Hybridisation(hybridization temperature=Tm probe –21ºC)
  1. dilute probe to 20 nM in hybridization buffer
  2. add 200ul hybridization mix per slide
  3. hybridize slides overnight covered with Nescofilm in a humidified chamber
Stringency Wash
  1. rinse in 2x SCC at hybridisation temperature
  2. 3 times 30 min in 50% formamide, 2xSSC at hybridisation temperature
  3. 5x 5 min in PBST at RT
Immunological Detection
  1. 1 h block in blocking buffer (2% sheep serum, 2mg/ml BSA in PBST) at RT
  2. o/n antibody incubation (1:2000 anti-DIG-AP Fab fragments in blockingbuffer) in a humidified chamber at 4ºC
  3. wash 5-7 times 5 min in PBST
  4. wash 3 times 5 min in AP buffer
COLOUR REACTION (RT, darkness)
  1. light sensitive colour reaction (NBT/BCIP) 1h-48h (400ul/slide) in a humidified chamber
  2. wash slides 3x 5 min in PBST
  3. mount in aqeous mounting medium (glycerol) or dehydrate and mount in Entellan.

    4.  

     

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
 
COLOUR 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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