Daniel:Notebook/RNAFISH/2014-11-22

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Revision as of 18:58, 24 November 2014 by >Djacobse (→‎Cell Fixation)
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Cell Fixation[edit]

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Trying RNA FISH again, this time using cell lines so I can play to my heart's content with conditions before I waste more tissue samples. I have cultured 4 cell lines, 3 new neuronal-based lines + NCI h1975. The new lines are U-87MG, T98-G, and BE2-C.

Slide Preparation (Dish)[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
  3. Allow cells to adhere for 6 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Rinse briefly in 1X PBS
  7. Incubate for 5 minutes in 1 mL of 2X SSCT
  8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
  9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm

I was on autopilot for this, and forgot that I use a slightly different fixation technique for tissue, but seeing as how these are all cultured cells, it shouldn't be a problem.