Matt:LabNotes/2015-12-14
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FISSEQ on Mouse Embryo Test 2[edit]
- Previous Test
- Try 2 samples: slide7 (16um thickness) and slide8 (20um thickness)
Embryo Section Info[edit]
- E7.5#3 slide1 is 16um (8 sections beginning of embryo + placenta) on the coverslip there is written A1, B1, C1, D1 to help for the orientation.
- E7.5#3 slide2 is 20um (8 sections beginning of embryo + placenta) on the coverslip there is written A2, B2, C2, D2 to help for the orientation.
- E7.5#3 slide3 is 25um (7 sections beginning of embryo + placenta) on the coverslip there is written A3, B3, C3, D3 to help for the orientation.
- E7.5#3 slide4 is 16um (7 sections mainly embryo) on the coverslip there is written A4, B4, C4, D4 to help for the orientation.
- Used: E7.5#3 slide5 is 20um (7 sections middle embryo) on the coverslip there is written A5, B5, C5, D5 to help for the orientation.
- Used: E7.5#3 slide6 is 25um (7 sections middle embryo) on the coverslip there is written A6, B6, C6, D6 to help for the orientation.
- E7.5#3 slide7 is 16um (10 sections middle embryo) on the coverslip there is written A7, B7, C7, D7 to help for the orientation.
- E7.5#3 slide8 is 20um (12 sections middle/end embryo) on the coverslip there is written A8, B8, C8, D8 to help for the orientation.
- E7.5#3 slide9 is 25um (6 sections end embryo+ adult liver) on the coverslip there is written A9, B9, C9, D9 to help for the orientation.
Protocol[edit]
- Previously did 0.01% Pepsin for 10min at RT and had too much tissue loss
- Try 0.01% Pepsin for 1-2min at 37C
Day 1[edit]
- Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
- When peeling off scotch tape from coverslip (that was used to tape coverslips to glass slides), it peeled off a clear film as well...
- What kind of clear film would develop on coverslip at -80C?
- Use Double-sided tape: Adhesives Research IS-8458-19
- Wash tissue section twice using cold nf-SSPE for 5min each
- A8 leaked so used 200 Gap filling glue
- 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 of cold 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
- Parafilm each dish and then put in plastic bag with wet tissue
Day 2[edit]
- Wash with 1X PBS twice
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component | Volume |
DEPC-H2O | 640 |
CircLigase Buffer 10X | 100 |
MnCl2 50mM | 50 |
Betaine 5M | 200 |
CircLigase II (100U/ul) | 10 |
Total | 1000 |
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Wash with 2X SSC + 30% formamide for 10min at 60C
- wash with 2X SSC, 1X SSC, 1X PBS once each
- Add 200ul RCA mix and incubate for 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT
- Wash with 2X SSC twice
- Image with Confocal
Image Results[edit]
- 20X 0.75 NA
A7 16um[edit]
File:MAX A7 16um Section1.jpg File:Composite A7 16um Section1.jpg
A8 20um[edit]
Section 1[edit]
File:MAX A8 20um Section1.jpg File:Composite A8 20um Section1.jpg
Section 2[edit]
File:MAX A8 20um Section2.jpg File:Composite A8 20um Section2.jpg