Daniel:Notebook/HiResChrPaint/2014-8-23

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Padlock Probes (Started 8/13/2014)[edit]

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Hybridization (From 8/21/2014)[edit]

  1. Remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
  2. Incubate in 2X SSCT at room temperature for 10 minutes
  3. Incubate in 0.2X SSC at room temperature for 10 minutes
  4. Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
  5. Add 20 uL DAPI + Slow Fade Gold to center of dish
  6. Cover with 22x22 #1.5 coverslip; seal with nail polish
    1. Allow at least 30 minutes for nail polish to dry
    2. Slides are quite stable for several months at 4C

Microscopy[edit]

Sample A[edit]

Sample A is stained with PPRL-488 (green) and Cot1-546 (red). The padlock probes should produce strong green signal in many cells, but there is only weak signal in a very small number of cells.

Sample B[edit]

Sample B is stained with Cot1-488 (green) and DMD-546 (red). The signal seems to be present in several cells, though it is difficult to determine, given the small scale of the signal (the probes target only a few kb). Zooming in to get only one or two cells helps detect the signal better. However, there does appear to be clear signal above the noise in #1 and #3. #2 is meant to show what most cells look like, containing strong Cot1 signal with very no signal from the DMD.