Matt:LabNotes/2015-6-23: Difference between revisions
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>Mzcai (Created page with "=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, permeabil...") |
>Mzcai m (→Procedure) |
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===Procedure=== | ===Procedure=== | ||
#Make PBT: 1X PBS + 0.1% Triton-X | #Make PBT: 1X PBS + 0.1% Triton-X | ||
#Rehydrate embryo #3 | #Rehydrate embryo #3 and #4 in 200ul PCR tube with attached caps | ||
#*75% MeOH in PBT for 10min rotating | #*75% MeOH in PBT for 10min rotating | ||
#*50% MeOH in PBT for 10min rotating | #*50% MeOH in PBT for 10min rotating | ||
#*25% MeOH in PBT for 10min rotating | #*25% MeOH in PBT for 10min rotating | ||
#*PBT for 10min rotating twice | #*PBT for 10min rotating twice | ||
#Only for embryo #4 | |||
* | #*Add 0.25% Triton-X in 1X PBS for 10min | ||
* | #*Add 0.01% Pepsin in 0.1N HCl for 5min | ||
#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 |
Revision as of 21:45, 25 June 2015
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 and #4 in 200ul PCR tube with attached caps
- 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
- Only for embryo #4
- Add 0.25% Triton-X in 1X PBS for 10min
- Add 0.01% Pepsin in 0.1N HCl for 5min
- 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