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
- They use 20X objective to image the whole 250um depth
ISH Protocol[edit]
- Protocol.pdf
- Fixed, dehydrated in methanol, and stored in -20C
- Rehydrated and permeabilized with ProK for ISH
Fixed samples[edit]
- File:MERT 2015-05-14 E7.5 FISSEQ.JPG
- Got 7 embryos each in a 2ml tube and stored in -20C
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]
- Make PBT: 1X PBS + 0.1% Triton X-100
- 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
- Wash with 1X PBS three times
- 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
- 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