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Scot's Notes from
CW-STED Sample-Prep Presentation
by Myriam Gastard (Leica), Jun 30 2010

Myriam has kindly provided key images/text PPT slides (as PDF) from her talk Jun 30 2010. If you use the images elsewhere, please give Myriam and Leica attribution and credit.

Contact information
(for additional details or guidance)
Myriam Gastard, PhD., Product Specialist
Tel: 866-830-0735, option3
Cell: 215-407-4488
(please be respectful, not too late at night!)
Addr: Leica Microsystems, Inc
410 Eagleview Blvd, Suite 107
Exton, PA 19341

Limited Amounts of Starter Reagents
(courtesy of Myriam/Leica)
Contact Miriam Wahl (MicFac)
Tel:410-614-6890 or 443-287-2859

Thiodiethanol (TDE, Sigma#88559), Excellent mounting medium

Alexa430-(goat α-mouse, Invitrogen) Ab (for 2-color)

Alexa430-(goat α-rabbit, Invitrogen) Ab (for 2-color)

Dylight488-(goat α-mouse, Rockland Immuno) Ab (best 1st color): outperforms Alexa488 for CW-STED

Scot's Notes from Seminar:

MANDATORY for super-resolution:

  • Use #1.5 coverslips ONLY (other side of sample can be glass slide)
  • Avoid DAPI and Quantum Dots (multiphoton effect from STED laser)
    NOTE: DAPI already in some mounting media (e.g. some versions of Prolong), so check first!

NEW INFO (from seminar; still refer to prior notes for 2-color sample prep and for 1-color sample prep):

  • CW-version of STED is still new, and so-called 'rules' are still being discovered. ONLY empiricism can confirm workability. Be willing to try new things with CW-STED!

  • Although many dyes work for confocal and can be imaged, the extra super-resolution by CW-STED demands extra stability of dyes/fluorescent proteins.

  • Live-cell work (1-color super-resolution; 25fps):
    • Prior advice against GFP wrong: some version eGFP will work. Original 'eGFP' from ~10 years ago are NOT as stable as more recent eGFP constructs.
    • Best to use mCitrine or mVenus.
    • AVOID phenol red in media.
  • 2-color super-resolution: To provide super-resolution, Alexa430 has trade-offs.
    • Only available as antibody conjugate.
    • Alexa430-antibodies are surprisingly weak (only available from Invitrogen/Molecular Probes). Best to incubate overnight, 4C.
    • As a single dye, don't expect to see Alexa430 by eye unless there's lots of labelling. It's really dim.
  • Mounting media suggestions (fixed cells/tissue):
    • If <30um specimen thickness: use Prolong (no DAPI!) + Antifade, but wait 2 days so cures to correct index of refraction {Moviol + Antifade works, too, but more of a pain to make}
    • If >30um specimen thickness: use 97% Thiodiethanol (TDE) + Antifade {Moviol + Antifade works, too, but more of a pain to make}.
      Protocol: Step up concentration of TDE by 15-30 min each in 25%, 50%, 75% and the 97% TDE. Only last step needs Antifade, but lots of pipetting to mix in the antifade (poor solubility in aqueous).
      Note: TDE will not solidify/cure and will remain liquid. Seal with nail polish and can use after dry.
  • Antifade suggestion: Start with 0.1% p-phenylenediamine (PPD, probably the most effective antifade)

Myriam's suggested starting protocol for 2-color:

  • Cells grow on # 1.5 coverslip
  • Cells fixed with PAF 4% 10 min., RT
  • Rinse 3x in PBS
  • Block with PBS/Triton X 0.2%/Normal goat serum 10%, 30 min in rotation at RT
  • Incubate in primary Ab in PBS/Tx/NGS 1 hr, RT. The first primary must have a stronger fluorescence compared to the second primary Ab.
  • Rinse 3x in PBS
  • Block for 30 min.
  • Incubate in secondary, Alexa 430, overnight, on rotation at 4 degree C.
  • Rinse, rinse, rinse!!!! At least 3x in PBS, in rotation at RT
  • Block in PBS/Tx/ normal serum (in accordance with the “second” secondary Ab species
  • Incubate in 2nd primary Ab for 1hr at RT
  • Rinse in PBS
  • Block
  • Incubate in DyLight 488, 1 hr, RT
  • Rinse in PBS
  • Mount in Prolong Gold (with antifade). Let cure overnight.
  • Enjoy the STED CW imaging!

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