Matt:LabNotes/2016-9-28

From ZhangLabWiki
Jump to navigation Jump to search

FISSEQ whole mount Mouse Embryo + Focus Clear[edit]

  • Previous experiment with PACT/CLARITY failed
  • This time try to do regular FISSEQ in mouse embryo like Matt:LabNotes/2016-3-8 and then use Focus Clear to image deeper into embryo
  • What this will tell us is whether there are rolonies in the mesoderm and endoderm
    • So far we have only seen rolonies in ectoderm and we don't know whether it is a probe diffusion/permeabilization issue or an imaging/light scattering issue

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. 4% PFA in PBS made 8/31/2016
    • 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
    • Sealed 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 ~15hr 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]

  • 3 Tries
    1. Add FISSEQ_Adpt_Cy3 and then add focusclear for 2 hrs
    2. Add FISSEQ_Adpt_Cy3 and then 5uM DRAQ5 and then add focusclear for 2 hrs
    3. Embed in 5% 199:1 polyacrylamide gel and then add FISSEQ_Adpt_Cy3
  1. Try 1
    • Preheat 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC to 75C
    • Add 100ul to embryo in tube and let sit for 10min at RT
    • Wash with 2X SSC twice
    • Add Focusclear and incubate at RT for 2hr
    • Mount on slide with Mountclear
  2. Try 2
    • Preheat 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC to 75C
    • Add 100ul to embryo in tube and let sit for 10min at RT
    • Wash with 2X SSC twice
    • Add 100ul 5uM DRAQ5 in 1X PBS and let sit for 15min at RT
    • Remove DRAQ5
    • Add Focusclear and incubate at RT for 2hr
    • Mount on slide with Mountclear
  3. Try 3
    • Embed in 5% 199:1 Acr:Bis Hydrogel on #1.5 Vectabond treated coverslip
      • Follow standard protocol, polymerize in 50uM spacer jig for 30min in Argon filled bag
    • Attach coverslip to bottom of dish
    • Wash with 1X PBS twice
    • Preheat 0.5uM FISSEQ_Adpt in 30% formamide + 2X SSC to 75C
    • Add 100ul to embryo in tube and let sit for 10min at RT
    • Wash with 2X SSC twice
    • IMAGE
    • Add Focusclear and incubate at RT for 2hr
    • IMAGE

Results[edit]

  • Try 3 first time imaging is positive control because no focusclear added yet
    • Result is normal, rolonies are bright obvious and numerous
  • Try 3 after adding Focusclear for ~30min
    • Embryo is harder to see by naked eye, less white
    • Rolonies are dim and hard to identify at normal gain, have to increase gain 600->750 and laser% 1->2
    • Looking for rolonies at deeper layers of cells show none, rolonies only in outermost layer of cells
  • Try 1
    • Rolonies are dim, have to increase gain 600->750 and laser% 1->2
  • Try 2
    • Almost no rolonies in Cy3
    • DRAQ5 does not look usual, small punctate signal more like rolonies than nuclei
    • Also dim signal so had to increase gain to 750 and layer% 5->10

Conclusion[edit]

  • Focusclear after Cy3 hybridization dims Cy3 signal
  • Focusclear does make tissue much more transparent
  • Rolonies do not exist past outermost layer of cells, need to permeabilize tissue more