Matt:LabNotes/2015-6-23

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Test Mouse Embryo Pre-Processing Protocol for DARTFISH

  • 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

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

Fixed samples

FISSEQ

  • 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

  1. Make PBT: 1X PBS + 0.1% Triton-X
  2. Rehydrate embryo #3
    • 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
  • At this point go into 0.25% Triton-X and 0.01% Pepsin?
  • Or do ISH protocol and do 6% hydrogen peroxide, ProK, and fix again...