multicolor by sequential staining

Approach Objective and Adventages

  1. Avoid some of the incompatiability issues of protein-DNA staining:
    • Protein stains in previously untreated tissue should always work
    • DNA stains should still work after protein imaging — it’s just the formamide treatment tends to kill the protein/antibody signal.
  2. Allows better multi-channel drift correction
    • multi-color images don’t easily work with feducial tracking, since its hard to find beads that don’t bleach and aren’t too bright that simultaneously work for all channels. Image-based drift correction is not as good, and is especially difficult between channels.
    • Red beads should work well with all 647 imaging
  3. No need for chromatic warping
  4. Everything can be imaged in Cy5. Avoids higher background/lower signal/reduced switching of other chromatic options.

Feducials

  • Important for precise registration of sample
  • should be a bonus for drift correction, if we modify the code to ID the feducials and use these for drift-correction

Test experiment

  • label sample with AATAT probe
  • Wash out probe (heat and then 60C in formamide for a few hours should do it).
  • Image blank probe
  • Restain with AATAT probe
  • Reimage (hopefully feducials still stuck). See if we get better alignment than 750, cy5, BX-C.
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