Leica CW-STED: Sample Prep Living up to Life STED CW: Sample Preparation -tubulin,(Rockland Immuno, Inc.). M. Gastard, Leica Microsystems, Inc. (personal communication) Myriam C. Gastard, PhD www.leica-microsystems.com 1 Living up to Life STED: Spectral Conditions Excitation STED Detection Band Requirements for STED CW: • Dye excitable with Argon laser • Still significant absorption at STEDc • No Excitation at STEDc 350 400 450 500 550 600 650 www.leica-microsystems.com Myriam Gastard, 10_0630 2 1 Leica CW-STED: Sample Prep Living up to Life 1 and 2-color STED CW Secondary Antibodies 1-color STED CW + = Best Good Fair DyLight 488 Alexa 488 FITC Chromeo 488 Atto 488 Oregon Alexa 430 (only in combination with DyLight, Chromeo, Alexa or Atto 488 and only with 2-color STED CW) 2 -color STED CW www.leica-microsystems.com Living up to Life 2-color STED CW Good Fluorescence Proteins Best Depending on the amount of the protein of interest, the brighter being the better. mCitrine, mVenus (514 nm excitation) eYFP (514 nm excitation) 3 DO NOT USE No DAPI eGFP (488 nm excitation) mCerulean (458 nm excitation) eCFP (458 nm excitation) www.leica-microsystems.com Myriam Gastard, 10_0630 4 2 Leica CW-STED: Sample Prep Living up to Life STED CW: Resolution -tubulin,(Rockland Immuno). M. Gastard, Leica Microsystems, Inc. (personal communication) Myriam C. Gastard, PhD www.leica-microsystems.com Measured Resolution confocal 5 Living up to Life STED average lateral resolution FWHM measured on fluorescent beads (n =30; confocal/ 60;STED) Confocal: STED: www.leica-microsystems.com Myriam Gastard, 10_0630 260 nm 64 nm 6 3 Leica CW-STED: Sample Prep Living up to Life Resolution Enhancement by STED FWHM 65 nm FWHM < 65nm FWHM ~260nm FWHM ~260nm confocal STED STED STED STED STED STED STED STED STED STED STED STED STED STED STED STED A threefold improved resolution can make 9 spots out of 1 ! 7 7/1/2010 www.leica-microsystems.com Page 7 Living up to Life Resolution: Reality checked by STED Do NOT expect to have a resolution below 100 nm if the protein(s) or structure of interest are bigger than 100 nm ! STED CW resolution (in red) vs confocal resolution (in green): AKTpS473 (Rockland Immuno, Inc, PA) M. Gastard, personal communication www.leica-microsystems.com Myriam Gastard, 10_0630 8 4 Leica CW-STED: Sample Prep Living up to Life STED CW: Sample Preparation Cells or tissue? Coverslip #1.5 or glass bottom dishes Fluorescence (1or 2-color) Tissue thickness? Chosen protocol? Mounting media? Sample thickness is > 30m Sample thickness is < 30m • Prolong + Antifade • TDE + Antifade • Moviol + Antifade • Moviol + Antifade www.leica-microsystems.com 9 Living up to Life STED CW: Thiodiethanol preparation for tissue mounting (TDE, Sigma, #88559) 6 Well-plate TDE 25% TDE 50% TDE 75% TDE 97% + Antifade • 15-30 minutes incubation at each step (depending on the sections thickness). • Mix VERY WELL (pipette in and out) the antifade with the TDE 97% prior to mount the tissue sections (eliminate the bubbles by centrifugation). • Use nail polish to keep the #1.5 coverslip in place for the night. • Seal the coverslip edges the following the morning with nail polish. www.leica-microsystems.com Myriam Gastard, 10_0630 10 5 Leica CW-STED: Sample Prep Living up to Life STED CW: Antifading reagents • P-phenylenediamine (PPD): Probably the most effective antifade (0.1-0.01%). • N-propyl gallate (NPG, 2%): Not very soluble; non-toxic and can be used on live cells. • DABCO (2.5%): Not as effective as the PPD, but is less toxic. www.leica-microsystems.com 11 Living up to Life STED CW: 2-color protocol (This is just an example and we are encouraging you to stay as close as possible to your usual protocol. Only adjust the “Must Know” to your protocol). • 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! www.leica-microsystems.com Myriam Gastard, 10_0630 12 6