TCEP Buffer
- 0.2M Tris pH 9 (200 uL Tris 1M pH 9.0)
- 1 mM Methyl viologen (10 uL of 0.1 M stock. Currently make fresh from powder. Toxic) (25.7 mg / mL)
- 1 mM Ascorbic Acid (10 uL of 0.1 M stock. Currently make fresh from powder.) 17.6 mg/mL
- 25 mM TCEP (50 uL of a 0.5 M stock vial. $5/vial, use fresh, no more than 1 day).
- 1% Glox (usual strength. 10 uL in 1 mL)
- 5% glucose
- ddH2O — fill to 1000 uL.
TCEP Buffer information
- See publication for more details
- 750 in TCEP 2800 photon (compared to 700 in BME and 3-400 in MEA).
- Allows for ~equal photon yield 750 and 647 imaging.
- Can image both at 200 Hz.
- not as robust — concentration of TCEP matters, laser power effects duty cycle (higher laser power = lower duty cycle. necessary for dense imaging)
- 750 gas laser causes vibrations that errode ability to see ‘hollow’ structure of microtubules (in any channel)
- 897 ultra camera runs at 60 Hz in 512×512
- not compatible with other dyes (e.g. Cy3B).
Standard STORM buffer
- 80 uL Dilution Buffer
- 20 uL of 50% Glucose
- 1 uL of BME
- 1.5 uL of GLOX solution
Components:
- Dilution Buffer
* 50 mM NaCl
* 10-200 mM Tris, pH 8.0
* (higher molar Tris recommended for longer STORM imaging)
- Oxygen Scavenger (GLOX)
* 200 uL of Dilution Buffer (see above)
* 14 mg Glucose Oxidase (Sigma Aldrich)
* 50 uL of catalase (20 mg/mL; Sigma Aldrich)
* Dissolve glucose oxidase in Dilution Buffer, vortex to mix. After mixing the catalase suspension well, add catalase to glucose oxidase solution.
Centrifuge at maximum speed for 1 min. Catalase may visibly precipitate out and remain at the bottom of the tube. Use the yellow supernatant for imaging buffers.
* Aliquot and freeze immediately. Store working tube at 4C for up to 1 week. Fresh (non-frozen) aliquots good for up to 2 weeks at 4C.
- 50% (by mass) Glucose stock
- BME (Sigma-Aldrich 63689-25ML-F)