Matt:LabNotes/2016-9-28
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FISSEQ whole mount Mouse Embryo + Focus Clear
- Previous experiment with PACT/CLARITY failed
- This time try to do regular FISSEQ in mouse embryo like Matt:LabNotes/2016-3-8 and then use Focus Clear to image deeper into embryo
- What this will tell us is whether there are rolonies in the mesoderm and endoderm
- So far we have only seen rolonies in ectoderm and we don't know whether it is a probe diffusion/permeabilization issue or an imaging/light scattering issue
Procedure
- Embryo is fresh and sitting in 1X PBS in 200ul PCR tube
Day 1
- Sterilize bench and tweezers with EtOH, RNaseZap
- 4% PFA in PBS made 8/31/2016
- 10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O
- Sealed 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