Revision as of 01:48, 5 February 2016 by >Mzcai
FISSEQ on Mouse Embryo Test 2
Embryo Section Info
Protocol
Day 1
- Prepare 2 plastic culture dish with 18mm hole, tweezers, large plastic dish, and 50C hot plate
- Use 1 week old 4% PFA at RT
- Take out fresh frozen mouse embryo sections from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Both dishes leaked and required glue
- Wash with cold nf-H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times
- Prepare Reverse Transcription Mix on ice and add
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
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (10ul BS(PEG)9 stock + 490ul 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
- 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
Imaging Results
- Position of sections are marked with a black sharpie on glass slide
File:MouseE7.5 Slide 20160127.jpg
Imaged slide A1 on 2-1-2016
- Positions labeled [row]_[column]
- Imaged with 20X 1um z step size
1-1
1-2
1-3
1-4
2-1
2-2
2-3
2-4
40X
- 1um z step size
- 0.35um z step size (system optimized)
63X
Imaged slide A2 on 2-3-2016
- Hybridized FISSEQ_Adpt 48 hours ago (2-1-2016) took some images, and then stripped and rehybridized fresh
1-1
- Guessing the first section was here and then mostly degraded
1-2
1-3
1-4