Matt:LabNotes/2016-3-8

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FISSEQ whole mount Mouse Embryo[edit]

Procedure[edit]

  • Embryo is fresh and sitting in 1X PBS in 200ul PCR tube

Day 1[edit]

  1. Sterilize bench and tweezers with EtOH, RNaseZap
  2. Make fresh 4% PFA in PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
    • Add Argon gas and seal with parafilm to keep it fresh longer
  3. Aspirate PBS from tube and add 200ul 4% PFA
    • Incubate at 37C for 15min
  4. Wash twice using cold nf-SSPE
  5. Add 0.25% TX-100 in nf-2XSSPE for 5min at RT
  6. Wash with cold nf-H2O twice
  7. Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C
  8. Wash with 2ml nf-PBS three times
  9. Prepare 2X 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 ~18hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  5. Wash with 1X PBS twice
  6. Add RNase H mix and incubate 1hr at 37C
    • H2O - 168ul
    • RNase H Buffer - 20ul
    • Riboshredder - 2ul
    • RNase H - 10ul
  7. Wash with nuclease-free H2O twice
  8. Add CircLigase mix and incubate 3hr at 60C
    • H2O - 128ul
    • CircLigase Buffer 10X - 20ul
    • MnCl2 50mM - 10ul
    • Betaine 5M - 40ul
    • CircLigase II 100U/ul - 2ul
  9. Wash with 1X PBS twice
  10. Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  11. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  12. Prepare RCA reaction mix on ice
    • H2O - 174ul
    • Phi29 Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • Phi29 DNA polymerase 100U/ul - 2ul
  13. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  5. Wash with 1X PBS twice

Imaging[edit]

  1. Add 66ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C
  2. Incubate for 10min at RT
  3. Wash with 2X SSC twice
    • Lost 2 embryos, only the largest embryo (#18) left
  4. Mount onto glass slide with 120um spacer between coverslip
    • Use microscope mounting medium
  • 552 Laser 1%
  • Cy3 Gain: 550
  • Tilescan 4x5 tiles with 10% overlap
  • To image whole embryo with max resolution (2k x 2k with 0.3um step size) takes ~5hr

Images[edit]

  • Tilescan image taken with 63X objection and 512x512 resolution, single z-plane

File:63X 512x512 Tilescan Merged z0 ch01.jpg

  • Image of anterior pre-crescent taken with 63X and 0.3um z step size over 100um (339 z slices)
    • Images not shown here
  • Tilescan image whole embryo taken with 63X and 1um z step size over 100um
    • The anterior pre-crescent as well as cells deeper in embryo show lack of rolonies
      • Guessing this is due to poor permeabilization

File:Projections63X 512x512 1umZstep Tilescan Merged-0001.jpg File:Projections63X 512x512 1umZstep Tilescan Merged-0008.jpg File:Projections63X 512x512 1umZstep Tilescan Merged-0012.jpg File:Projections63X 512x512 1umZstep Tilescan Merged-0015.jpg File:Projections63X 512x512 1umZstep Tilescan Merged-0018.jpg

Conclusion[edit]

FISSEQ Process[edit]

  • Using fresh embryo this time has better results than fixed sections
  • Need better permeabilization
    • Rolonies are only at the outer cells of embryo

Whole Mount Process[edit]

  • Need to flatten embryo more so a single focal plane contains more information
  • Need a method that allows stripping/hybridizing dye-probes
    • Embed embryo in PA gel? How to embed and flatten