Matt:LabNotes/2015-9-2

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Imaging BA8 with DARTFISH suppv2 for 3D Decoding[edit]

Sample[edit]

  • I think this sample has suppv2 because in ConfocalStore Hosuk imaged sample on 5-18-2015 and named it "S2_V4_Supp2_2015-05-18"
  • Notes on lid of culture dish:
      1. 2 5/16/15
    • BA8 (4/29/15)
    • Fix: 4% Form/37C/15min
    • 0.25% TX100/10min/RT
    • 0.01%Pepsin/5min/37C
    • RT:+adUTP 5/16/15
    • 15min@RT->37C
    • RCA: 5/18/15

Adding Fiducial Beads[edit]

  • Sonicate 1:2000 diluted fluorescent spheres in bath sonicator
  • Wash sample with 10nM HEPES
  • Add beads to sample and incubate 5min at RT
  • Wash sample with 10nM HEPES twice
  • Add BS(PEG)9 for 1hr at RT
  • Wash with 1X PBS twice
  • Add 1X Tris pH 8.0 for 30min at RT
  • Wash with 1X PBS twice

Imaging on 9-2-2015[edit]

  • Cycle00: Fiducial Beads + FISGA_Adpt_Cy3
  • Cycle01-Cycle05
    • Leica software crashed before Cycle05 due to trying to save Cycle04 when there was no more disk space left
      • Had to re-find positions
  • 4 positions
    • 0.3um z step size
    • 48 zSlices