Matt:LabNotes/2015-7-6

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Test Mouse Embryo Pre-Processing Protocol for DARTFISH[edit]

ISH Protocol[edit]

  • Protocol.pdf
    • Fixed, dehydrated in methanol, and stored in -20C
    • Rehydrated and permeabilized with ProK for ISH

Fixed samples[edit]

FISSEQ[edit]

  • To save padlock probes will test fixation/permeabilization sample conditions with FISSEQ
  • Will use universal probe for all rolonies (FISSEQ_Adpt Cy3?) to check
  • To image, sandwich embryo between glass slide and coverslip

Procedure[edit]

  1. Make PBT: 1X PBS + 0.1% Triton X-100
  2. Rehydrate embryo #7 in 2ml original tube
    • 75% MeOH in PBT for 10min rotating
    • 50% MeOH in PBT for 10min rotating
    • 25% MeOH in PBT for 10min rotating
    • PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge tube
  3. Wash with 1X PBS three times for both tubes
  4. Make RT Mix (2X volume) on ice
    • H2O - 314ul
    • M-MuLV RT Buffer 10X - 40ul
    • dNTP 25mM- 4ul
    • aa-dUTP 2mM- 8ul
    • FISSEQ_RT primer (nonamer) 100uM - 10ul
    • RNase Inhibitor 40U/ul- 4ul
    • M-MuLV RTase 100U/ul- 20ul
  5. Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C
  6. Wash with 1X PBS once
  7. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  8. Wash with 1X PBS twice
  9. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  10. Wash with 1X PBS twice
  11. Add RNase H mix and incubate 1hr at 37C
    • H2O - 168ul
    • RNase H Buffer - 20ul
    • Riboshredder - 2ul
    • RNase H - 10ul
  12. Wash with nuclease-free H2O twice
    • ASPIRATED EMBRYO! :'(