Matt:LabNotes/2016-7-19

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Revision as of 22:24, 19 July 2016 by >Mzcai (→‎PACT Protocol)
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CLARITY and PACT for FISSEQ in Mouse Embryo E7.5

  • Need more permeabilization to get enzymes and reagents deeper into tissue
  • To permeabilize will remove lipids with detergent in long incubation
  • Need to embed tissue in hydrogel matrix to create structure that will survive detergent

Experiment

PACT Protocol

  1. Fix embryo in 4% PFA at 37C for 15min
  2. Make 4mL PACT solution on ice
    • 4% acrylamide in 1X PBS plus 0.25% VA-044 (degassed)
    • 3.584mL MilliQ H2O + 0.448mL 10X PBS + 0.448mL 40% acrylamide (cold) + 0.0112g 0.25% VA-044 (cold)
  3. Wash with 1X PBS
  4. Incubate embryo in solution at 4C overnight (multiple days?)
  5. Polymerize gel by incubating at 37C for 3hr
  6. Make 500mL 8% SDS solution
    • 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
  7. Remove embryo from gel
    • Roll on kimwipe to remove bits of gel
  8. Incubate in 8% SDS at 37C on rotator for overnight (multiple days?)
    • Tissue will not be fully clear until put into refractive index matching media
  9. Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator (2 days?)
    • Need to wash away SDS or it will precipitate forming cloudy spots

CLARITY Protocol

  1. Make 1mL CLARITY solution on ice
    • 4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed)
    • 4mL 40% PFA + 1mL 40% 19:1 acrylamide:bis + 0.1g VA-044 + 4mL 10X PBS + 31mL H2O
    • Can vary the acrylamide/bis-acrylamide concentrations if multiple embryos
  2. Incubate embryo in solution at 4C overnight
  3. Polymerize gel by incubating at 37C for 3hr
  4. Make 500mL 8% SDS solution
    • 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
  5. Remove embryo from gel
    • Roll on kimwipe to remove bits of gel
  6. Incubate in 8% SDS at 37C on rotator for overnight (multiple days?)
    • Tissue will not be fully clear until put into refractive index matching media
  7. Wash embryo in PBS + 0.1% TX-100 at 37C on a rotator (2 days?)
    • Need to wash away SDS or it will precipitate forming cloudy spots