Matt:LabNotes/2015-10-20
Jump to navigation
Jump to search
DARTFISH on BA8 Sections
- Try two samples: one without suppressor oligos and one with suppv2
- If sample is good use suppv2 sample to compare with Gwen's RNAscope images of BA8 with 4 genes: OLFM1, PDE1A, SCL17A7, RELN
Protocol
Day 1
- Prepare 2 plastic culture dishes with 12x17mm rectangle hole, tweezers, large plastic dish, and 50C hot plate
- EtOH then UV
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O
- Take out BA8 sections (2 coverslips) from -80C and incubate on 50C hot plate for 3min
- Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X PBS by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 10min (normally 5min) at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with cold 1X PBS three times
- Prepare 2X Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C