Matt:LabNotes/2015-11-11

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20X Decoding to Compare with RNAscope

Finding Position to image

Big Plan

  • 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

  • Top right of tissue: (68.87, 69.21)
  • Bottom right of tissue: (68.87, 53.72)
  • 9 positions imaged = 4.5mm

Decoding Procedure

  • Used 100ul 0.5uM dcProbe1 through 5
  • Cycle00
  • Cycle01...Cycle05
  • DRAQ5
    • Added 100ul 5uM DRAQ5 to tissue and incubated 15min at RT
    • No wash steps, went straight to imaging