Tzahor Lab

Double Flourescent

Double fluorescent In situ hybridization for paraffin section

♦ DAY 1

Preparation of slides for hybridization

  • Use DEPC treated solutions for all steps prior to hybridization!!!
  • Defrost ~200ml PFA 4% at the beginning
  • Better to use parafilm as cover slips

Paraffin slides

  • Bake slides on hot plate at 60°c, 10'
  • Allow slides to reach RT
  • Dewax in xylene 2 x 15'
  • Rehydrate to PBS :
    • 100% EtOH 2 x 5'
    • 75% EtOH 5'
    • 50% EtOH 5'
    • 25% EtOH 5'
    • PBS 2 x 5'
  • 4% PFA 10' (Don't throw the PFA!)
  • PBT Rinse
  • PBT - 2 x 5'
  • {If you get a lot of background – do the H2O2 stage from day 2 now (2% in PBT)}
  • PK (Roche) in PBS - 5μg/ml (stock is 10mg/ml – put 100μl in 200ml PBS) - 10', 37°c or 30' RT
  • PBT - 2 x 5'
  • 4% PFA 5' (Re-use PFA from first post-fix)
  • PBT Rinse
  • PBT - 2 x 5'
  • Acetylation 10' :

Add 625μl acetic anhydride to 250ml 0.1M triethanolamine (TEA). Prepere the 0.1M solution by adding 3.3 TEA to 246ml of DDW and shake well-use immediately!)

  • PBT Rinse
  • PBT - 2 x 5'
  • Rinse H2O DEPC
  • Warm the hybe solution to 65°c
  • Air dry slides ~10-30'
  • Turn on the Oven (62°-65°c)
  • Add about 3μl of probe to 97μl prewarmed hybe solution.

Put 100μl on each slide and cover with parafilm.

  • Place slides in humidified slide box (use Whatman paper or paper towels soaked in 5 x SSC/50% formamide)
  • Incubate O.N at 62°-65°c (depend on the background you get)

♦ DAY 2

Post-Hybridization Washes
* Pre-warm wash solutions

  • Remove cover slips and rinse in 5 x SSC
  • 1 x SSC / 50% formamide 30' at 65°c
  • TNE 10' at 37°c (Don't throw this TNE)
  • TNE + RNase A(Roche)- 20μg/ml (stock is 1mg/ml – dilute x 50 ) - 30', 37°c
  • TNE 10' at 37°c (Re-use TNE from first wash)
  • 2 x SSC 20' at 65°c
  • 0.2 x SSC 20' at 65°c
  • 0.2 x SSC 20' at 65°c

Antibody Incubation and Detection

  • MABT - 2 x 5' at RT
  • 2% H2O2 in TNT (stoke is 30%) 30' at RT
  • TNT 5' at RT
  • Blocking with TNB-buffer 45' - 1h at RT (200μl TNB per slide, cover with parafilm)
  • Detection of the digoxigenin-labeled probe – α Dig POD 1:250 in TNB, 100μl per slide, cover with parafilm. 4°c O.N (if you are doing a single staining and not a double one or if you like to work for very long hours, you can leave it for ~30' at RT and finish the protocol in 2 days instead of 3)

♦ DAY 3

* Don't expose the slides too much to light after you add the Cys. Cy2 goes bleaching very fast – remember that when you are taking pictures. if you are doing a single staining – use Cy3

  • TNT -  3 x 5' (shake)
  • Staining – first centrifuge the Cy3 (or Cy2) 1' ,14000rpm
  • Cy2 (or Cy3) 1:100 in amplification reagent (TSA signal amplification kit, Perkin Elmer Life Sciences, Cat.No. NEL760), 100μl per slide, cover with parafilm. 15'-20'
  • 2% H2O2 in TNT (stoke is 30%) 5' at RT – quenching the HRP from the first staining.
  • TNT -  3 x 5' (shake)
  • Detection of the fluorescein-labeled probe – α Fluorescein POD 1:150 in TNB, 100μl per slide, cover with parafilm. 30' at RT.
  • TNT -  3 x 5' (shake)
  • Cy3 (or Cy2) 1:100 in amplification reagent (TSA signal amplification kit, Perkin Elmer Life Sciences, Cat.No. NEL760), 100μl per slide, cover with parafilm. 15'-20'
  • 2% H2O2 in TNT (stoke is 30%) 5' at RT
  • TNT -  3 x 5' (shake)
  • DAPI 1:10,000 in TNT, 10' at RT
  • TNT -  1 x 5' (shake)
  • Elvanol (put the elvanol first in 55°c for 10')