Sample Preparation for STED CW 1 and 2-color ST E D

STimulated Emission Depletion (STED)
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Sample Preparation for STED CW 1 and 2-color
ACTIN
MICROTUBULES
Confocal
Confocal
STED
STED
Myriam Gastard, PhD, Application and Tech Support, Exton, PA, 19341.
www.leica-microsystems.com
03/14/2011
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Resolution Enhancement by STED
confocal
FWHM 65 nm
FWHM < 65nm
FWHM ~260nm
FWHM ~260nm
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 !
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3/16/2011
Resolution: Reality check by STED
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STED CW resolution (in red) vs confocal resolution (in green): AKTpS473 (Rockland Immuno, Inc, PA)
REMEMBER
Do NOT expect to have a resolution below 100 nm if the protein(s) or structure of interest
are bigger than 100 nm !
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1-color STED CW
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STED: Spectral Conditions
Excitation
STED
Detection Band
Requirements for STED CW:
• Dye excitable with Argon laser
• Still significant emission at λSTEDc
• No Excitation at λSTEDc
350
400
450
500
550
600
650
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2-color STED CW
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What is the challenge?
458
488
592 depletion laser
Shift stock
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1 and 2-color STED CW
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Secondary Antibodies
1-color STED CW
• DyLight 488: gave the highest resolution, bright.
• Oregon green 488: very bright, be careful with precipitates.
• Chromeo 505: Bright and allows better separation.
• Alexa 488 , Alexa 514: not as bright or stable than the previous ones.
2 -color STED CW
in combination with one dye from the single color STED CW list
• Atto 425, antibody or Streptavidin conjugated.
• BD Horizon V500, Streptavidin conjugated.
• Pacific Orange, antibody conjugated.
• NBD-X (Succinimidyl).
Do Not Use: DAPI, QDOTs, or other fluorescent dyes which are not excitable
with the Argon laser.
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2-color STED CW
Recommended pair for 2-color STED CW
Channel 1
Channel 2
Comment
V500 (Strep)
+
Oregon Green 488/Chromeo
505
Strongly recommended, best
combination so far (V500 staining via
secondary biotinilated antibody biotin
+ streptavidin)
No dye separation required
V500 (Strep)
+
Any green dye, reported to work
for STED CW
No dye separation required
Pacific Orange (Secondary
Antibodies conjugated)
+
DyLight488 / Alexa488
(excluding Oregon Green 488
and Chromeo 505)
Dye separation required
Atto 425 (Anti-mouse)
+
DyLight 488, Chromeo 505,
Oregon Green 488, Alexa 488
To be used with protein or structure
of interest bigger than 100 nm due to
a lower efficiency of the dye in STED
CW.
No dye separation required.
NBD-X
+
Any other green dye reported to
work for STED CW
No commercial conjugates for NBD-X
so far
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2-color STED CW
Fluorescent Proteins
458 nm excitation
488 nm excitation
514 nm excitation
eCFP
eGFP
eYFP
mCerulean
Emerald
mCitrine, mVenus
NBDx
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STED CW: “Must Remember”
Cells or tissue?
Coverslip #1.5 or glass bottom
dishes
Fluorescence (1or 2-color)
Coverslip # 1.5
Chosen protocol?
Mounting media?
Sample thickness is < 30µm
• Prolong kit (Not Prolong Gold)
• Moviol + Antifade
Sample thickness is > 30µm
• Prolong kit (Not Prolong gold)
•TDE + Antifade
• Moviol + Antifade
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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 the antifade (pipette in and out) with the TDE 97% prior to mount the tissue
sections (eliminate the bubbles by centrifugation).
• Use nail polish (high quality uncolored nail polish) on the #1.5 coverslip corner to keep it in
place for the night (must be kept flat).
• Seal the coverslip edges the following morning with nail polish.
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2-color STED CW
Mounting Media
Best
Good
Do Not Use
Prolong kit + Antifade
Mowiol + Antifade
(will polymerize)
(will polymerize)
Slowfade
Thiodiethanol
(TDE, Sigma, #88559). Add
Antifade (will stay
visceous)
Vectashield
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STED CW: Antifading reagents
• DABCO (2.5%): Not as effective as the PPD, but is less toxic.
• Or you can also use the proprietory antifade from the Prolong kit, without DAPI.
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STED CW: 2-color protocol
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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, (or methanol, 5-7 min, -20 ºC).
• 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.
• Rinse 3x in PBS.
• Block for 30 min.
• Incubate in secondary: Pacific Orange, Atto 425, V500, 1 hours at RT or on rotation at 4 degree C overnight.
PS: For the Horizon V500 labeling: biotinylated antibody for 1 hour at RT followed by Horizon V500 Streptavidin, 1 hr at RT.
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 3x in PBS.
• Rince once in tap water.
• Mount in Prolong Kit (or TDE + antifade).
• Let cure at least overnight (at best 48 hrs to reach the maximum RI).
• Keep the slide at 4 degree C and protected from the light.
• Enjoy the STED CW imaging!
Myriam Gastard, PhD, personal communication, Leica Microsystems, Inc. USA
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Examples of optimization of the acquisition for selected dyes pairs,
+
Dye1
Name
Excitation
Emission
Pacific Orange
458
535 - 580
Pacific Orange
458
Horizon V500
Dye2
Name
Excitation
Emission
+
DyLight 488, Alexa 488 / Any 488
(excluding Oregon Green 488)
488
496
500 – 530
505 - 530
500 -580
+
Any 488 (excluding Oregon Green 488)
488
496
514
500 – 580
505 – 580
525 - 580
458
465 – 500
465 - 510
+
Oregon Green 488, DyLight 488 / any 514
dyes
488
514
505 - 580
520 - 580
NBD-X
458
535 – 580
Or
500 - 580
+
488
500 – 530
Or
500 -580
Atto 425
458
465 - 510
+
514
520 - 580
DyLight 488, Alexa 488 / Any 488
(excluding Oregon Green 488)
Oregon Green / Any 488 / Any 514 dyes
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Selected spectra
ATTO 425
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ATTO 425 (Ex / Em)+ DyLight 488 (Ex / Em)
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STED CW: Sample Preparation
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Question?
Need any help before / during sample preparation?
Please contact me directly at: 866-830-0735, Option 3
or on my cell phone (given directly during the talk).
Myriam Gastard, PhD, Application and Tech Support, Exton, PA, 19341.
03/14/2011
www.leica-microsystems.com
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