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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== *[[Media:ChiLab_Embryo_ISH_Protocol.PDF | 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 | 450px]] **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-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 <!--#Wash with 1X PBS twice #Add RNase H mix and incubate 1hr at 37C #*H2O - 168ul #*RNase H Buffer - 20ul #*Riboshredder - 2ul #*RNase H - 10ul -->
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