Daniel:Notebook/RNAFACS/2015-9-30

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Revision as of 23:27, 1 October 2015 by >Djacobse (→‎Nuclei Adhesion)
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Nuclei Adhesion[edit]

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Nuclear Adhesion Protocol Basis

First thing is first, I want to make sure that my protocol for nuclear adhesion is a valid one. Since I won't be using any probe for this, I'll stick to the mouse nuclei. Once I am sure of the efficacy, I can try once with human nuclei to make sure they work as well. Most papers I've seen require fixed nuclei, and since I am not using fixed nuclei, we'll start with that.

  1. Clean glass slide in 100% EtOH; allow to air dry
  2. Remove cells from -80C and spin down for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  3. Add 500uL of 4% paraformaldehyde to the tube and wait 5 min
  4. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  5. Add 500 uL of 70% EtOH
  6. Centrifuge cells for 5 min at 1000g
    1. Remove supernatant, careful not to disturb the pellet
  7. Resuspend nuclei in 20 uL 70% EtOH
  8. Deposit on to glass slide
  9. Use a coverslip to gently spread the 20 uL across the slide
    1. Be careful not to scratch the slide
  10. Dry the slide using a heating block at 60C for 10-15 min
  11. Add 15 uL Slowfade + DAPI and seal with a coverslip

This worked very well. There were plenty of nuclei annealed everywhere within the circle. I will repeat the experiment using human nuclei tomorrow