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]

  1. Prepare 2 plastic culture dishes with 12x17mm rectangle hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH then UV
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O
  3. Take out BA8 sections (2 coverslips) from -80C and incubate on 50C hot plate for 3min
  4. Submerge coverslips in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X PBS by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add cold 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 10min (normally 5min) at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with cold 1X PBS three times
  11. 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
  1. Incubate 10min at 4C and then ~18hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. 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
  1. Add mix to sample and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~24hr

Day 3[edit]

  1. Wash with 1X PBS twice
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate at RT for 30min
  5. Wash with 1X PBS twice
  • To sample with suppv2
  1. Sonicate 1:4000 diluted fluorescent spheres in bath sonicator
  2. Wash sample with 10mM HEPES
  3. Add beads to sample and incubate 5min at RT
  4. Wash sample with 10mM HEPES twice
  5. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) and incubate at RT for 1hr
  6. Wash with 1X PBS twice
  7. Add 1X Tris pH 8.0 for 30min at RT
  8. Wash with 1X PBS twice
  • To both samples
  1. Add 200ul 0.5uM FISGA_Adpt in 2X SSC + 30% Formamide pre-heated to 75C and incubate for 10min at RT
  2. Wash with 2X SSC twice
  3. 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]

  1. 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
  1. 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