Matt:LabNotes/2016-1-27
FISSEQ on Mouse Embryo Test 2[edit]
- Previous test using sections of fixed embryo
- This time try fresh frozen mouse embryo sections
Embryo Section Info[edit]
Protocol[edit]
Day 1[edit]
- Prepare 2 plastic culture dish with 18mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- 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[edit]
- 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[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
Imaging Results[edit]
- 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[edit]
- Positions labeled [row]_[column]
- Imaged with 20X 1um z step size
1-1[edit]
File:MAX A1 1-1 20X.jpgFile:A1 1-1 20X z15 ch01.jpg
1-2[edit]
File:MAX A1 1-2 20X.jpgFile:A1 1-2 20X z17 ch01.jpgFile:Composite A1 1-2 20X.jpg
1-3[edit]
File:MAX A1 1-3 20X.jpgFile:A1 1-3 20X z17 ch01.jpg
1-4[edit]
File:MAX A1 1-4 20X.jpgFile:A1 1-4 20X z16 ch01.jpg
2-1[edit]
File:MAX A1 2-1 20X.jpgFile:A1 2-1 20X z21 ch01.jpg
2-2[edit]
File:MAX A1 2-2 20X.jpgFile:A1 2-2 20X z19 ch01.jpg
2-3[edit]
File:MAX A1 2-3 20X.jpgFile:A1 2-3 20X z17 ch01.jpg
2-4[edit]
File:MAX A1 2-4 20X.jpgFile:A1 2-4 20X z18 ch01.jpg
40X[edit]
- 1um z step size
- 0.35um z step size (system optimized)
File:MAX A1 2-4 40Xoptimized.jpgFile:A1 2-4 40Xoptimized z46 ch01.jpg
63X[edit]
File:MAX A1 2-4 63X.jpgFile:A1 2-4 63X z052 ch01.jpg
Imaged slide A2 on 2-3-2016[edit]
- Hybridized FISSEQ_Adpt 48 hours ago (2-1-2016) took some images, and then stripped and rehybridized fresh (rehybridized images shown here)
1-1[edit]
- Guessing the first section was here and then mostly degraded
File:MAX A2 1-1 40X.jpgFile:A2 1-1 40X s0 z21 ch01.jpg
1-2[edit]
File:MAX A2 1-2 40X.jpgFile:A2 1-2 40X s0 z48 ch01.jpg
1-3[edit]
File:MAX A2 1-3 40X.jpgFile:A2 1-3 40X s0 z38 ch01.jpg
1-4[edit]
File:MAX A2 1-4 40X.jpgFile:A2 1-4 40X s0 z41 ch01.jpg
Discussion[edit]
The following emailed to Paola:
- All the sections have a thin layer of tissue inside the ring. So the rings are ~20um thick but the tissue inside the ring is <5um thick. This middle has a lot of rolonies too.
- Noticeable difference between A1 and A2. All the A1 sections stayed on the glass and showed much more rolonies. I'm wondering if this is a difference between your sectioning technique or how I fixed them.
- One section was in direct contact with metal of 37C incubator while the other was not during fixation.
- How do you think the tissue looks? Maybe you remember which order you cut them in and can relate that to their result.
Signal 48hr post hybridization vs immediately post hybridization[edit]
20X vs 40X vs 63X Objectives[edit]
- Used Plot3D.m and plotcube.m in MATLAB to find some random rolonies and show how many zSlices the rolony can be detected in
- Sample: A1_2-4
- Used system optimized z-step size unless indicated otherwise
20X[edit]
- Other rolonies also about ~6 z-steps high
- Equivalent to ~6um
- Rolonies aren't actually 6um long, just can be detected because poor z-resolution
40X[edit]
z-step = 1um File:A1 2-4 40X 3dplot.png
40X system optimized z-step[edit]
z-step = 0.35um File:A1 2-4 40Xoptimized 3dplot.png