Matt:LabNotes/2015-6-23

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

  • For project using DARTFISH on mouse embryo to study cell lineage in cardiac development
  • They normally fix, permeabilize, and hybridize probes all in tube suspension (whole mount ISH)
    • They use 20X objective to image the whole 250um depth
      • Since we used 63X objective with 1.4 NA we probably cannot do 250um depth and will require sectioning

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 #3 and #4 in 200ul PCR tube with attached caps
    • 75% MeOH in PBT for 10min rotating
    • 50% MeOH in PBT for 10min rotating
      • Embryo may have split in two? Maybe TX-100 concentration was too high in the 50% MeOH solution and needs to be remade
    • 25% MeOH in PBT for 10min rotating
    • PBT for 10min rotating twice
  3. Wash with 1X PBS three times
  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 embryos and incubate for 10min at 4C and then incubate overnight at 37C
  • In tubes there is white fibrous precipitate? No way to distinguish embryos from this white stuff (too cloudy and lots of white debris)
    • Looks like gDNA but there's too much to have been from the embryo
    • List of chemicals in M-MuLV enzyme and buffer: Tris-HCl, NaCl, EDTA, DTT, NP-40, glycerol, KCl, MgCl2
    • List of chemicals in RNase Inhibitor: Hepes-KOH, KCl, DTT, glycerol