Matt:LabNotes/2015-8-17

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BA8 Section DARTFISH suppv2 with Fiducial Beads[edit]

  • 3-4 month old BA8 tissue section
  • suppv2 design
  • PFA frozen from Matt:LabNotes/2015-8-4
  • 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]

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Thaw 5ml 4% PFA at RT
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
    • New solution made by Justin
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
    • There was significant degradation after this step, only a sliver of tissue left
  10. Wash with nuclease-free PBS three times
  11. 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
  1. Incubate 10min at 4C and then ~18hr at 37C
  2. Wash with 1X PBS once
  3. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
  4. Wash with 1X PBS twice
  5. Add Tris pH8.0 and incubate 30min at RT
  6. Wash with 1X PBS twice
  7. RNA Removal
    • Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
    • Even the sliver of tissue has lost more
  8. Wash with 1X PBS twice and then store in 1X PBS parafilmed
    • Put in 4C box labeled "RNA FISH and DARTFISH Cultured Neurons"