Matt:LabNotes/2015-8-4
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BA8 Section DARTFISH with Fiducial Beads
- Try again: Matt:LabNotes/2015-8-3
- with fresh PFA
- Practice DARTFISH on BA8 tissue sections
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
DARTFISH on BA8
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Made 40ml 4% PFA
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
- 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
- Still had leak so used gap-filling adhesive 200 to seal
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- 0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed
- 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
- Wash with nuclease-free PBS three times
- Can already see tissue is degrading
- 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 ~15hr at 37C
- Tissue is degrading too easily and PFA shouldn't be the problem
- Maybe 0.25% TX-100 has higher concentration than labeled because of evaporation, will try making new one
- Or skip pepsin digestion?
- Could it be due to long storage time?