eSTORM Project

Sample preparation protocol

Ranbel Sun and Drago Guggiana-Nilo

Solutions:

Actin Buffer:

100 mM PIPES (MW 302.37)

10 mM EGTA (MW 380.4)

5 mM MgSO4 (MW 120.4)

0.3 M Mannitol (MW 182.17)

2% (w/v) Glycerol (MW 92.09)

Preparation (50 ml):

Dissolve 1.5 g of PIPES and 0.19 g of EGTA into about 15 ml of water. The powders won’t actually dissolve until the solution is titrated to pH 7 with 0.4 M NaOH (around 25-30 ml). The titration has to be done with strong agitation, and while doing it add 0.03 g of MgSO4 and 3 g of Mannitol. Both should dissolve easily. Finally, after the titration is completed, transfer the solution to a 50 ml Falcon tube and weight 1 g of Glycerol in the cap of the tube. Then proceed to cap the tube with it and mix the solution until the glycerol is dissolved. Open the tube (some solution might drip a bit after the cap is opened) and complete it’s volume to 50 ml with water.

Fluorescent dye:

0.66 uM Alexa 647 - Phalloidin (MW 1950)

Stock:

Alexa647-Phalloidin reconstitution: dissolve the lyophilized contents of the tube (300 units) in 1.5 ml of methanol on ice to yield a final concentration of 200 units/ml, or the equivalent of 6.6 uM (2). Cover the tube in tinfoil when done to protect from photodamage.

Imaging buffer:

- TN Buffer

50 mM Tris-HCl (MW 157.56)

10mM NaCl (MW 58.44)

10% (w/v) Glucose (MW 180.16)

Preparation (50 ml):

Dissolve 0.39 g of Tris-HCl in 35 ml of water and adjust pH to 8 using NaOH (pH starts at around 5 and it takes about 5 ml of 0.4M NaOH to bring it to 8). Then add 5 g of Glucose, 0.03 g of NaCl and complete volume to 50 ml. Store at 4 C.

- GluOx

50 mM Potassium Phosphate Buffer

0.5 mg/ml Glucose Oxidase

40 ug/ml Catalase

Prepare a 1M solution of Potassium Phosphate Buffer (pH 7.0) by dissolving 0.11 g of K2HPO4 and 0.05 g of KH2PO4 into 10 ml of water. Dilute 20-fold to get the desired 50 mM.

Preparation (1ml):

Dissolve Dissolve 50 mg of Glucose Oxidase and 139 ul (4 mg) of Catalase in 50 mM Potassium Phosphate Buffer (pH 7.0, complete volume to 1 ml using approximately 850 ul of buffer) inside a 1.5 ml Eppendorf tube. Store at 4 C.

- BME

143 mM beta-Mercaptoethanol (MW 78.13 , d = 1.114 g/ml)

pH adjustment:

NaOH (MW 40.00)

HCl (MW 36.46)

Procedure:

1) Cut a clean, square and thin slice of onion epidermis

2) Incubate for 1 hr in 495 ul of actin buffer with glycerol and add 5 ul of the Alexa647-Phalloidin stock in an 1.5 ml Eppendorf in the dark.

3) After incubation, transfer the onion slice to a new 1.5 ml Eppendorf tube containing 1 ml of imaging buffer (980 ul of TN Buffer, 10 ul of GluOx and 10 ul of BME )and incubate for 5 minutes, also in the dark.

4) Mount the slice in a glass slide along with 10 ul of the imaging buffer and top with a coverslip.

5) Image the sample.

References:

1) Lab Ref

2) Invitrogen Phalotoxins manual

3) Olyslaegers and Verbelen

4) Dempsey, Vaughan, Chen, Bates and Zhuang