Matt:LabNotes/2016-6-15

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FISSEQ whole mount embryo[edit]

  • Objective:
    • Test multiple harsher permeabilization conditions
    • Embed in thin (~50um) PA hydrogel for imaging (FISH and stripping)

Procedure[edit]

  • Embryos are 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 TX-100 in nf-2XSSPE for 5min at RT
    • 0.5% for 10, 15, 20, 20min at RT while on orbital shaker
  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 5min 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 ~16hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (16ul stock BS(PEG)9 + 784ul 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
  1. Mount in 50um thick 5% 199:1 acrylamide:bis hydrogel on coverslip
    • Embryo 1 got smeared
    • Embryo 2 glass cracked
    • Only embryo 4 (20min incubation) made it through 100%
  2. Attach coverslip to bottom of dish

Imaging[edit]

  1. Add 200ul 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
  4. Added 200ul 5uM DRAQ5 (diluted in PBS from 100uM brown tube Hosuk made) to tissue and incubated 15min at RT


  • 552 Laser 1% Cy3 Gain 550
  • 638 Laser 5% Gain 800
  • Images not shown here because they aren't as good as the images from 2nd session
    • The first z-slice starts in middle of embryo so misses some sections of embryo nearest to the glass
  1. Replace buffer with 1X PBS and stored in 4C over the weekend

Imaging 2nd Time[edit]

  • After the weekend replaced 1X PBS with 200ul 5uM DRAQ5 and incubated another 15min at RT
  • 552 Laser 2% Gain 600
  • 638 Laser 5% Gain 800
  • Tilescan 3x5 tiles with 10% overlap
  • 512x512 pixels for fast imaging time
  • 1um z-step size, 96um total thickness

Images[edit]

File:20160620 WME FISSEQ 20minTX100 3Dprojection 0degree.jpg
File:20160620 WME FISSEQ 20minTX100 3Dprojection 30degree.jpg
File:20160620 WME FISSEQ 20minTX100 3Dprojection 40degree.jpg
File:20160620 WME FISSEQ 20minTX100 3Dprojection 45degree.jpg

Conclusion[edit]

Imaging Notes[edit]

  • In the future need to use smaller z-step size

Flatten Embryo in Gel[edit]

  • Able to flatten embryo to ~100um in gel
    • Gel is probably polymerized at 50um but swells veritically to ~100um
    • Not sure what the effects of flattening the embryo has on rolonies
      • The act of squeezing the embryo between glass surfaces applies a nonzero shear force on embryo that led to tearing in the other embryos
      • Even slight shear force may damage walls of embryo
  • Flattening embryo should make total area near glass larger so even if we can't image as deeply we could be getting more information
    • As long as we can see into mesoderm layer

Permeabilization[edit]

  • Hard to tell if increased permeabilization now there are rolonies in second layer (mesoderm)
    • Not being able to see rolonies in the walls of embryo makes it hard to distinguish layers
    • Based on DRAQ5 nuclei staining for glass-side of embryo the second cell layer is zslices 9-16
    • In zslices 9-16 there are a few rolony signals, this is a positive indication that permeabilization helps
  • Paola reported not being able to detect signal past top layer in ISH staining and DAPI
  • CLARITY may be our best/only option