slides 2012

CHECK THIS

LLCPk-1

coverslips, fixed in 3.5% formaldehyde and stored in PBS-tween-azide in coplin jars in the refrigerator:

  • 30 unmounted,

  • 10 mounted, stained only with DAPI

  • 20 stained for tubulin, and mounted with DAPI

  • 20 stained for actin and tubulin, and mounted with DAPI

  • 10 serum starved, stained for tubulin, mounted with DAPI

    • for Lab 4.1
    • Grow first in regular medium (otherwise they won't stick)
    • Replace medium with serum free medium.
    • Fix after 24 hours
  • 10 Arrested with 100nM nocodazole (8 - 16 hours)

    • for Lab 4.1
    • Treat with nocodazole late in the day
    • Fix the next day in para-glut or formaldehyde
    • Stain for tubulin, mount with DAPI

Nocodazole stock is 33 mM in DMSO
Make 100 µL 1 mM: 3 µL (33mM) + 97 µL medium
Make 1 mL 6µM: 6 µL (1mM) + 994 µL medium
Add 1 drop (~50 µL) 6µM to each dish of ~3 mL

OR

Make 30 mL 100 nM: 3 µL (1mM) in 30 mL medium
Change the medium in each dish.

3t3

12 for Lab 3.3, unmounted