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]
- 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 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 ~18hr 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]
- Add 66ul 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
- Lost 2 embryos, only the largest embryo (#18) left
- 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
- The anterior pre-crescent as well as cells deeper in embryo show lack of rolonies
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