Matt:LabNotes/2015-10-12
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DARTFISH on BA8 Sections
- Try one sample with second fixation step after permeabilization with TX-100 and Pepsin
- If sample is good use 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 17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and RNase Free sterilize, then rinse with copious amounts of MilliQ water
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free 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
- One glass cracked so only one sample left DO SECOND FIXATION
- 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 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with cold 1X PBS three times
- Add 500ul 4% PFA for 10min at RT and
- 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
Day 2
- Wash with 1X PBS once
- Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
- Wash with nf-H2O twice
- Prepare Ampligase mix on ice
- Preheat H2O + Padlock probes + suppv2 Oligos to 85C and then snap cool before adding Ampligase buffer and enzyme
Component | Volume |
DEPC-H2O | 20 |
Ampligase Buffer | 10 |
Justin's Batch 5 7/22/2015 Padlock Probes 320nM]] | 31.25 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr