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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
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