Matt:LabNotes/2015-8-17
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BA8 Section DARTFISH suppv2 with Fiducial Beads[edit]
- Similar to Matt:LabNotes/2015-8-4 but with suppressor oligos and 5 cycle decoding
- 3-4 month old BA8 tissue section
- suppv2 design
- PFA frozen from Matt:LabNotes/2015-8-4
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
- Try only 5min incubation with TX-500 to see if that decreases tissue degradation
DARTFISH on BA8[edit]
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Thaw 5ml 4% PFA at RT
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- New solution made by Justin
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- There was significant degradation after this step, only a sliver of tissue left
- Wash with nuclease-free PBS three times
- Prepare Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Wash with 1X PBS once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add Tris pH8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Even the sliver of tissue has lost more
- Wash with 1X PBS twice and then store in 1X PBS parafilmed
- Put in 4C box labeled "RNA FISH and DARTFISH Cultured Neurons"