Matt:LabNotes/2015-7-7

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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 #6 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
    • Accidentally aspirated embryo again
    • This form factor won't work for DARTFISH, too unreliable for exchanging solutions and will not work for 5 cycles of dye hybridization/stripping