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
- They use 20X objective to image the whole 250um depth
ISH Protocol
- Protocol.pdf
- Fixed, dehydrated in methanol, and stored in -20C
- Rehydrated and permeabilized with ProK for ISH
Fixed samples
- File:MERT 2015-05-14 E7.5 FISSEQ.JPG
- Got 7 embryos each in a 2ml tube and stored in -20C
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
- Make PBT: 1X PBS + 0.1% Triton-X
- 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...