Biology Reference
In-Depth Information
Reagents
Bleach Solutions
1. Bleach solution:
a. 0.4 mL bleach
b. 0.4 mL 10N KOH
c. 3.2 mL water
2. Fix/permeabilization solution:
a. 200
m
L 10% PFA
b. 10
m
L 10% Triton X-100 (Sigma) or 5
m
L 10 mg/mL lysolecithin in chloro-
form (Sigma). Lyoslecithin is much gentler than Triton, and will preserve fine
structure, but also results in less overall extraction, leading to higher back-
ground signal in some cases. For preserving fine filaments in the epidermis,
lyoslecithin is preferable.
c. 48
m
L 0.5 M PIPES (pH = 6.8)
d. 25
m
L 0.5 M HEPES (pH = 6.8)
e. 1
m
L 1 M MgCl
2
f. 10
m
L 0.5M EGTA
g. 196
m
L ddH
2
O
3. Phalloidin solution:
a. 6
m
L 6.6
m
M Alexa-488
b. 114
m
L1
PBS
Other solutions:
1. 10% PFA
a. 1 g paraformaldehyde
b. 30
m
L 5N NaOH
c. 10 mL 60 mM PIPES (1.2 mL 0.5M PIPES + 8.8 mL ddH
2
O)
Incubate
30 min in 65
C water bath until PFA is dissolved (note: do not exceed
65
C!)
2. 10% Triton
a. 1 mL Triton X-100
b. 9 mL ddH
2
O
Place on a nutator platform to mix thoroughly
3. 0.5 M PIPES
4. 0.5 M HEPES
5. 1 M MgCl
2
a. Dissolve 203.3 g of MgCl
2
6H
2
O in 800 mL of dH
2
O. Adjust the volume to
1 L with dH
2
O. Dispense into aliquots and sterilize by autoclaving or filtering.
Keep aliquots closed when they are not being used.
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