Matt:LabNotes/2016-7-19

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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 (20hrs)
  5. Polymerize gel by incubating at 37C for 3hr
    • Did not degas under Argon gas but can do next time if not polymerized enough
  6. Make 500mL 8% SDS solution
    • Could use 0.2 M boric acid or 0.01 M PBS (1X)
    • 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
    • 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
  7. Remove embryo from gel
    • Pipette away surrounding hydrogel and wash with 1X PBS
  8. Incubate in 8% SDS at 37C on rotator for overnight
    • 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 for 6 hrs
    • Need to wash away SDS or it will precipitate forming cloudy spots

CLARITY Protocol

  1. Make 4mL CLARITY solution on ice
    • 4% PFA, 0.95% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044, 1X PBS (degassed)
    • 0.5mL 40% PFA + 0.125mL 40% 19:1 acrylamide:bis + 0.0125g VA-044 + 0.5mL 10X PBS + 3.875mL H2O
  2. Incubate embryo in solution at 4C overnight (20hrs)
  3. Polymerize gel by incubating at 37C for 3hr
  4. Make 500mL 8% SDS solution
    • Could use 0.2 M boric acid or 0.01 M PBS (1X)
    • 6.183g boric acid, 200mL MilliQ H2O, pH with NaOH to pH=8.5, MilliQ H2O to 300mL total, then add 200mL 20% SDS
    • 50mL = 5mL 10X PBS + 20mL 20% SDS + 25mL H2O
  5. Remove embryo from gel
    • Pipette away surrounding hydrogel and wash with 1X PBS
    • Roll on kimwipe to remove bits of gel
  6. Incubate in 8% SDS at 37C on rotator for overnight
    • 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 for 6 hrs
    • Need to wash away SDS or it will precipitate forming cloudy spots

FISSEQ

  • tdTomato positive embryos were taken by Paola for confocal/two-photon microscopy test
  • For tdTomato negative embryos do FISSEQ

Day 1

  1. Wash with PBS three times
  2. Prepare 6X Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM FISSEQ_RT 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~16hr at 37C