Matt:LabNotes/2016-9-28
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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]
- Sterilize bench and tweezers with EtOH, RNaseZap
- 4% PFA in PBS made 8/31/2016
- 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
- Sealed with parafilm to keep it fresh longer
- Aspirate PBS from tube and add 200ul 4% PFA
- Incubate at 37C for 15min
- Wash twice using cold nf-SSPE
- Add 0.25% TX-100 in nf-2XSSPE for 5min at RT
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C
- Wash with 2ml nf-PBS three times
- 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 |
- Incubate 10min at 4C and then ~15hr at 37C
Day 2[edit]
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
- Add RNase H mix and incubate 1hr at 37C
- H2O - 168ul
- RNase H Buffer - 20ul
- Riboshredder - 2ul
- RNase H - 10ul
- Wash with nuclease-free H2O twice
- 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
- Wash with 1X PBS twice
- Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- 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
- Add RCA mix and incubate at 30C overnight (~15hrs)
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 200ul 1M Tris (pH 8.0) for 30min at RT
- Wash with 1X PBS twice
Imaging[edit]
- 3 Tries
- Add FISSEQ_Adpt_Cy3 and then add focusclear for 2 hrs
- Add FISSEQ_Adpt_Cy3 and then 5uM DRAQ5 and then add focusclear for 2 hrs
- Embed in 5% 199:1 polyacrylamide gel and then add FISSEQ_Adpt_Cy3
- 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
- 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
- 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
- Embed in 5% 199:1 Acr:Bis Hydrogel on #1.5 Vectabond treated coverslip
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