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)
- Last time realized no signal past outermost layer of cells
- Hoping more permeabilization will allow rolony reaction to occur throughout embryo
Procedure[edit]
- Embryos are fresh and sitting in 1X PBS in 200ul PCR tube
Day 1[edit]
- Sterilize bench and tweezers with EtOH, RNaseZap
- 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
- Aspirate PBS from tube and add 200ul 4% PFA
- Incubate at 37C for 15min
- Wash twice using cold nf-SSPE
- Add TX-100 in nf-2XSSPE for 5min at RT
- 0.5% for 10, 15, 20, 20min at RT while on orbital shaker
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 5min 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 ~16hr at 37C
Day 2[edit]
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (16ul stock BS(PEG)9 + 784ul 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
- 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%
- Attach coverslip to bottom of dish
Imaging[edit]
- Add 200ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C
- Incubate for 10min at RT
- Wash with 2X SSC twice
- 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
- 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