Matt:LabNotes/2016-6-15: Difference between revisions
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*552 Laser 1% | *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==== | |||
*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, 92um total thickness | |||
==Images== | ==Images== | ||
==Conclusion== | ==Conclusion== | ||
===Gel Embedding=== | ===Gel Embedding=== | ||
* | *Able to flatten embryo to ~50um in gel | ||
===Permeabilization=== | ===Permeabilization=== | ||
Revision as of 18:46, 27 June 2016
FISSEQ whole mount embryo
- 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
- Embryos are fresh and sitting in 1X PBS in 200ul PCR tube
Day 1
- 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
- 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
- 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
- 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
- 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, 92um total thickness
Images
Conclusion
Gel Embedding
- Able to flatten embryo to ~50um in gel