Matt:LabNotes/2015-7-6: Difference between revisions

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#*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
#**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
#*25% MeOH in PBT for 10min rotating
#*PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge tube
#*PBT for 10min rotating twice and add PBT to a second empty 2ml centrifuge tube

Revision as of 23:32, 6 July 2015

Test Mouse Embryo Pre-Processing Protocol for DARTFISH

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-100
  2. Rehydrate embryo #7 in 2ml original tube
    • 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 and add PBT to a second empty 2ml centrifuge tube
  3. Wash with 1X PBS three times for both tubes
  4. 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
  5. Add 200ul RT Mix to embryo tube and empty tube and incubate for 10min at 4C and then incubate overnight at 37C