In situ Hybridisation with Dig labelled probes

 see also:   http://www.fruitfly.org/about/methods/RNAinsitu.html

RNA probes (with the Boehringer kit)

  1. prepare labelling mix:
          1 µl DNA (linearised, 1 µg)
          2 µl 10x NTP+Dig labelling mix 
          2 µl 10x transcription buffer 
          2 µl RNA polymerase (40 U of T7, T3, or SP6)
          1 µl RNase inhibitor (20 U)
          12 µl water (or ad 20 µl final volume) 
  2. incubate 2 h at 37°C 
  3. optional: add 2 µl DNaseI (RNase free), incubate 15 min at 37°C 
  4. add 0.8 µl 0,5M EDTA, 2 µl 5M LiCl, 75 µl Ethanol (-20°C), mix, leave at 4°C for longer than 30min 
  5. spin at 4°C for 10 min, wash with 70% Ethanol (4°C) 
  6. dissolve in 98 µl DEPC-water, add 2 µl 5M NaOH, incubate at room temperature                                      t(in min) = (Lo - Lf)/k/Lo/Lf, Lo: original length of RNA, Lf: 0,075 kb, k: 0,11/kb min, for a 1,5 kb fragment t is 115 min 
  7. add 20 µl 10M NH4Acetat and 240 µl Ethanol, incubate >30 min, spin 10 min, wash, dry (pellet may not be visible) 
  8. dissolve pellet in 20 µl DEPC water, store at -20°C, typical yield: 10 µg RNA
  9. mix 2 µl of probe with 1 µl tRNA (50 mg/ml), 20 µl water boil 4 min at 100°C, cool down rapidly in ice water, dry ice/EthOH, or liquid N2,   add 200 µl of hybridisation solution


  in situ hybridisation of Drosophila embryos

  1. dechorinate embryos in 50% bleach, fix for 20 min in heptan/4% formaldehyde in PBS, pop in methanol, embryos may be stored at -20°C
  2. transfer fixed embryos to PBST, rinse 3x wash in PBST 2x, 5min 
  3. incubate 10 min in 1:1 hyb-sol/PBST 
  4. incubate 10 min in hyb-sol 
  5. prehybridise in hyb-sol for 1h at 57°C for RNA probes
  6. add probe in hyb-sol, incubate over night at 57°C   
  7. save probe (can be used several times), rinse 3x with hyb-sol (prewarmed) 
  8. wash 3x30 min in hyb-sol at 57°C
  9. (optional) wash as follows
    10 min 4:1 hyb-sol/PBST at 57°C
    10 min 3:2 hyb-sol/PBST at 57°C
    10 min 2:3 hyb-sol/PBST at 57°C
    10 min 1:4 hyb-sol/PBST at room temerperature
  10. wash 2x 20 min in PBST with 1% BSA at room temperature  
  11. rinse with PBST*

Detection with AP
  1. add digoxigenin antibody coupled with alkaline phosphatase (Boehringer, Fab Fragments) at 1:2000 in PBST*, incubate for 2 h at room temperature or over night at 4°C
  2. rinse 3x with PBST*
  3. wash 4x 15 min with PBST*
  4. wash 3x 5 min with AP buffer 
  5. start reaction to 1 ml of AP buffer add 4,5 µl NBT and 3,5 µl BCIP, incubation time may vary from few min to 3 h or even overnight, reaction should be kept in dark, but may be checked for colour development, e. g. string 30-60 min, frs 4 h 
  6. rinse 4x with PBST 
  7. go through an ethanol series to 100% ethanol, each step 10 min
  8. colour will change to blue and pink background will be washed out.
  9. go the ethanol series down to PBT and mount in appropriate media, like glycerol or aquapolymount. Before mounting in araldite, refix the stained embryos, because the stain might be lost in aceton wash.
Detection with POD/fluorescence
  1. add digoxigenin antibody couples with peroxidase at 1:200 (or less) in PBST*, incubate for 1-2h at room temperature or overnight at 4°C
  2. rinse 3x with PBST*
  3. wash 4x 15 min with PBST*
  4. prepare reaction solution: dilute TSA-Cy3 stock 1:200 in reaction buffer
  5. start the staining with 200 µl of reaction solution (adjust volume depending on amount of embryos), protect from light, develop for 1- 20 min depending on the probe (e. g. slam 1 min)
  6. stop staining reaction with 10 µl of 20% Na-azide or washing with PBST
  7. proceed with mounting or additional stainings (all protected from light)

hybridisation temperature: standard protocol  55°C for RNA probes, 45°C for DNA probes, to increase specificity the temperature may be increased to up to 65°C

digoxigenin antibody:  (stored at 4°C)
a-Dig-AP (alkaline phosphatase, Fab fragments, Roche 1093274): preadsorbed on wt embryos at 1:10, may be stored at 4°C for a year or longer (add azide), depending on the conditions the concentration may be increased (up to 1:500) or lowered (up to 1:5000)
a-Dig-POD (peroxidase, Fab fragments, Roche 1207733):  use at 1:200 in PBT (no azide!)

RNAase: degradation by RNAaseA may be a problem as long as the probe is not in hybridisation solution (with formamide).After hybridisation RNA degradation is no risk anymore, because RNAaseA does not degrade the RNA:probe duplex.

TSA-Cy3:  Perkin Elmer SAT 704A001EA
store at 4°C, together with buffer, protect from light

 

buffer:

  • NTP+Dig labelling mix (10x):  
    Dig-11-UTP: 10mM solution (25 µl, Roche #1209256)
      10 mM ATP
      10 mM GTP
      10 mM CTP
      6,5 mM UTP
      3,5 mM Dig-11-UTP, pH7,5

  • transcription buffer (10x), 
    for SP6, T3, T7 RNA polymerase, store at -20°C
      • 400 mM Tris-HCl, pH 8
      60 mM MgCl2
      100 mM DTT
      20 mM Spermidine
      100 mM NaCl

  • DEPC treatment: add 2 ml diethyl pyrocarbonate per 1 l of water/solution incubate at 37°C overnight and autoclave

  • PBST: DEPC treated PBS with 0,2% Tween

  • PBST*:  PBS with 0,2% Tween with additional 300mM NaCl, may decrease the background staining of AP, when long periods of incubation are required

  • NBT/BCIP:  NBT (nitrobluetetrazolium, Sigma), 75 mg/ml in 70% DMF, BCIP (X-phosphate, Sigma), 50 mg/ml in DM, store at -20°C

  • Hybridisation solution: 
     store at -20°C (else formamide hydrolyses)

    50% formamide
    		 25 ml
    5xSSC
    		 12,5 ml (20xSSC)
    50 µg/ml heparin
    		 50µl (50 mg/ml)
    0,2% Tween
    		 500 µl (10%)
    100 µg/ml tRNA
    		 100 µl (50 mg/ml)
    water
    		 ad 50 ml

  • AP buffer: 
    AP buffer forms a precipitate after some time

    100 mM NaCl
    		 1 ml (5 M)
    50 mM MgCl2
    		 2,5 ml (1 M)
    100 mM Tris pH9.5
    		 5 ml (1 M)
    0,2% Tween
    		 500 µl (10%)
    dd water
    		 ad 50 ml