Matt:LabNotes/2015-11-11

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20X Decoding to Compare with RNAscope[edit]

Finding Position to image[edit]

Big Plan[edit]

  • Try to match one of the same locations that Gwen took tile images with RNAscope
    • Her tile images are 5.6mm long but since this BA8 has some tissue degradation I don't need to image as much
  • Try to find a strip as long as possible
  • Use 20X objective for large FOV (584um x 584um)
  • Use x-step size of 500um so 84um overlap between positions can be used to stitch them together

Reference Positions using Stage Coordinates[edit]

  • Top right of tissue: (68.87, 69.21)
  • Bottom right of tissue: (68.87, 53.72)
  • Start position to match Tile 2 of Gwen's images (66.86, 62.02)
    • This is 2mm from the straight edge of tissue section, approximately same as Gwen's
    • About 7mm from the top of tissue, approx same as Gwen's Tile 2
  • End position (62.86, 62.02)
    • This where tissue integrity has reached a reasonable limit worth imaging
    • This makes total imaged distance 4.5mm
  • 9 positions imaged = 4.5mm

Decoding Procedure[edit]

  • Used 100ul 0.5uM dcProbe1 through 5
  • Cycle00
  • Cycle01...Cycle05
  • DRAQ5
    • Added 200ul 5uM DRAQ5 (diluted in PBS from 100uM brown tube Hosuk made) to tissue and incubated 15min at RT
    • No wash steps, went straight to imaging
      • 5% 638nm laser Gain of 650
  • Something odd happened to the tissue in positions 1-4 (you can see the transition in position 4)
    • It ruins the morphology and makes nuclei hard to distinguish
    • Also took images directly adjacent to positions 1-4 to substitute for nuclei counts