Matt:LabNotes/2015-8-18
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BA8 Section DARTFISH suppv2 with Fiducial Beads[edit]
- Repeat Matt:LabNotes/2015-8-17 but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation
- 3-4 month old BA8 tissue section
- suppv2 design
- 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
- Made 40ml 4% PFA at RT
- 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
- 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 0.01N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- 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
- Wash with H2O twice
- Add 100nM Padlock Probe Mix with 1000X suppv2 oligos and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr
- Wrapped with parafilm to prevent evaporation
Component | Volume |
DEPC-H2O | 20.18 |
Ampligase Buffer | 10 |
Padlock Probes 320nM | 31.25 |
100nM suppv2 Oligos | 28.57 |
Ampligase | 10 |
Total | 100 |
- Wash with 1X PBS once
- 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 6hr at 30C
Component | Volume |
H2O | 172 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
2mM aa-dUTP | 4 |
Phi 29 (low conc) | 2 |
Total | 200 |
- Wash with 10mM HEPES
- Sonicate 1:2000 diluted beads for 3 minutes in sonicator bath
- Add 200ul to sample and incubate 5min at RT
- Wash with 10mM HEPES once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) 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 and store in 1X PBS at 4C overnight
Decoding[edit]
- Cycle00 = Fiducial beads (488) and FISGA_Adpt (Cy3)
- Cycle01 = dcProbe1
- Cycle02 = dcProbe2
- Cycle03 = dcProbe3
- Cycle04 = dcProbe4
- Cycle05 = dcProbe5
- Cycle06 = Repeat of Cycle01
- When aspirating there was little solution on the dish...
- did I forget to wash with 2X SSC before starting imaging?
- Is the room so hot that it evaporated in an hour?
- When aspirating there was little solution on the dish...
- 63X objective (184um x 184um FOV)
- z step size = 0.3 um
- z stack size = 50-51 zSlices
- 6 Positions
- Each position takes ~11min to image
- Each position takes ~10GB and is taxing on memory