Daniel:Notebook/RNAFACS/2015-9-30
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Nuclei Adhesion[edit]
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.
- Clean glass slide in 100% EtOH; allow to air dry
- Remove cells from -80C and spin down for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500uL of 4% paraformaldehyde to the tube and wait 5 min
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Add 500 uL of 70% EtOH
- Centrifuge cells for 5 min at 1000g
- Remove supernatant, careful not to disturb the pellet
- Resuspend nuclei in 20 uL 70% EtOH
- Deposit on to glass slide
- Use a coverslip to gently spread the 20 uL across the slide
- Be careful not to scratch the slide
- Dry the slide using a heating block at 60C for 10-15 min
- 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