Matt:LabNotes/2015-10-20
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DARTFISH on BA8 Sections[edit]
- 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[edit]
Day 1[edit]
- 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
Day 2[edit]
- 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) or (H2O + Padlock probes) to 85C and then snap cool before adding Ampligase buffer and enzyme
- With suppressor for more intact tissue
Component | Volume |
DEPC-H2O | 21 |
Ampligase Buffer | 10 |
Justin's Batch 7 8/31/2015 Padlock Probes 329nM]] | 30.43 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Without suppressor for more degraded tissue
Component | Volume |
DEPC-H2O | 20 |
Ampligase Buffer | 10 |
Justin's Batch 6 8/6/2015 Padlock Probes 166nM]] | 60 |
Ampligase | 10 |
Total | 100 |
- Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~24hr
Day 3[edit]
- Wash with 1X PBS twice
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- Add RCA mix and incubate 18hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 4[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
- Wash with 1X PBS twice
- To sample with suppv2
- Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
- Wash sample with 10mM HEPES
- Add beads to sample and incubate 5min at RT
- Wash sample with 10mM HEPES twice
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1X Tris pH 8.0 for 30min at RT
- Wash with 1X PBS twice
- To both samples
- Add 200ul 0.5uM FISGA_Adpt in 2X SSC + 30% Formamide pre-heated to 75C and incubate for 10min at RT
- Wash with 2X SSC twice
- Add 400ul 2X SSC for imaging
Imaging Results[edit]
- Image positions on tissue that match positions Gwen did tilescan on RNAscope samples
- I imaged positions X horizontal distance starting from side AB
- Started from a vertical position approx lined up with point C
- This means I started from deep in white matter and slowly moved in superficial direction towards gray matter
File:GwenRNAscope BA8 TileLocations.jpg
- Gwen's tiles are 5.6mm long and starting about 1.5-2mm from deep white matter (side AB)
- To match the tiles, I need to see rolonies ~7mm from side AB
- As seen in images below, significant degradation even 5mm from side AB
20151020_BA8_DARTFISH_V4[edit]
0mm[edit]
File:20151020 BA8 V4 BF Pos1 Zero.jpg File:20151020 BA8 V4 MAX Pos1 Zero.jpg
5mm[edit]
File:20151020 BA8 V4 BF Pos2 5mm.jpg File:20151020 BA8 V4 MAX Pos2 5mm.jpg
6mm[edit]
File:20151020 BA8 V4 BF Pos3 6mm.jpg File:20151020 BA8 V4 MAX Pos3 6mm.jpg
7mm[edit]
File:20151020 BA8 V4 BF Pos4 7mm.jpg File:20151020 BA8 V4 MAX Pos4 7mm.jpg
20151020_BA8_DARTFISH_V4_suppv2[edit]
0mm[edit]
File:20151020 BA8 V4suppv2 BF Pos1 Zero.jpg File:20151020 BA8 V4suppv2 MAX Pos1 Zero.jpg
4mm[edit]
File:20151020 BA8 V4suppv2 BF Pos3 4mm.jpg File:20151020 BA8 V4suppv2 MAX Pos3 4mm.jpg
5mm[edit]
File:20151020 BA8 V4suppv2 BF Pos2 5mm.jpg File:20151020 BA8 V4suppv2 MAX Pos2 5mm.jpg
Conclusion[edit]
- The superficial layers (gray matter) of the tissue degraded too much
- Based on BF images 4mm-7mm away from the white matter side the tissue didn't completely fall off glass (Vectabond good) but only very thin spotty layer remained
- Hosuk's previous sample (started 5/4/2015) did not have this issue nearly as bad
- Gwen suggested as the BA8 tissue is taken in and out of freezer for sectioning, freeze/thaw will inevitably weaken outer tissue layers
- Hosuk also tried a 90sec pepsin incubation (normally 5min) on one sample and it looks the best in terms of tissue retention
- Maybe try looking at white matter of RNAscope samples instead
- The rolony count is too low, even for the sample without suppressors and in white matter area where tissue did not degrade as much
- Could there be a problem with the enzymes?
- They were kept in -20C box near front so would be first to thaw when people open the freezer for long periods of time
- Around this time Circligase II stopped working: Week earlier vs Week later
- Moved enzymes to back of freezer and then to enzyme box and threw out old M-MuLV RTase
- Could it be due to tissue on coverslips having touched the back of other coverslips when someone moved the box in -80C?
- Secure coverslips to glass slides next time with tape and put in glass slide holder
- Could there be a problem with the enzymes?