3.3 2013 | ISB Server Wahoo

Submitted by kdorfman on Thu, 09/26/2013 - 14:40

M 9/30

Labeling Cellular Structures

Cells

paraglut fixed LL's for students to stain for tubulin (make extra!)
paraglut fixed LL's for students to stain for phalloidin
paraglut fixed 3t3's for students to stain for phalloidin
live LL's to fix in paraglut (2014: treat first with mitotracker, then fix in formaldehyde no detergent)

Solutions

Rat anti-tubulin

Each tube has 3 µL antibody

Add
250 µL PBS-Tw-Az
255 µL 2% BSA
=510 µL, enough for 10 reactions (50 µL per coverslip, 60 min 37C)

Goat anti-rat

Each tube has 3 µL antibody

Add
192 µL PBS-Tw-Az
195 µL 2% BSA
=390 µL, enough for 7 reactions (50 µL/coverslip 37C 30min)

Phalloidin

50 µL/ coverslip (15 min at RT)

2 coverslips per group

100 µL/group

aliquoted to make 100 µL. Directions on tube.

Mitotracker

diluted in serum-free non-CO2 medium

1 mL/group

100 nm working concentration

stock is 1 mM in DMSO

Mix 1 µL in 10 mL

Aliquot ~1.2 mL/group

Either view live, or fix in 3.2% formaldehyde (in PBS) NO DETERGENT

Materials