advantages of the alexafluor dyes - unicaen · 01 imaging issues and alexa fluor author:...
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Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Mount
Culture Cells
Microscopy
Non-specific Binding
PhotobleachingLow Signal Intensity
Workflow Problems
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Laser-scanningcytometryEL4 cells biotin-anti-CD44+ streptavidin conjugates
Alexa Fluor Dyes – Photo Stability
Photobleaching dye solutions in capillary tubes
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Alexa Fluor 488 vs Fluorescein Bleaching2x Real Time
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Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Mount
Culture Cells
Microscopy
Non-specific Binding
PhotobleachingLow Signal Intensity
Workflow Problems
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Alexa Fluor ® Conjugated Secondary Antibodies
Simply the Brightest
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Alexa Fluor ® 647 vs. Cy5AF
647
GAM
Compa
ny A
Cy5
GAM
Compa
ny B
Cy5
GAM
Compa
ny D
Cy5
GAM
Compa
ny E
Cy5
GAM
Compa
ny F
Cy5
GAM
Compa
ny C
Cy5
GAM
Compare Alexa Fluor conjugates to any other fluorescent conjugates
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Alexa Fluor ® : pH Insensitive
AF488
FITC
Oregon Green
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Colour Selection ♦ Brightness ♦ Photostability
Alexa Fluor ® Dyes across the spectrum
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Multiplexing-The Four Main Colours
Emission wavelengths: Blue green orange/red far red
Alexa Fluor ® 594Texas Red, Cy3.5
Alexa Fluor ® 647Cy5, APC
Alexa Fluor ® 555Rhodamine,TAMRA, TRITCCy3
Alexa Fluor ® 488Fluorescein (FITC)Cy2
Alexa Fluor ® 350Coumarin, AMCA
350 400 450 500 550 600 650 700
DAPI/UV FITC TRITC FAR RED
Colour Selection ♦ Brightness ♦ Photostability
350 488 555 647
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Tools & Accessories
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Protecting the fluorescence signal -Antifade Reagents
Prolong Gold ®
•Optimized formulation-Superior antifade with little or no impact on initial fluorescence intensity
•Ready to use - dropper bottle
•Cures to a firm gel – no more nail polish
NEW: SlowFade Gold ® - antifade mounting media which doesn’t polymerise
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Fix cells
Permeabilise
Blocking Buffer
Primary Antibody
Secondary Antibody
Nuclear Stain
Mount
Culture Cells
Microscopy
Non-specific Binding
PhotobleachingLow Signal Intensity
Workflow Problems
| Life Technologies Proprietary & Confidential | 14
Increase S/N ratio - Non-specific background staining
Problem�non-specific binding of dye conjugates
�standard blocking agents don’t work
Solution•Image-iT TM FX Signal Enhancer – non-serum based blocking reagen t
Decrease background - increase specific signal
Non-specific binding - any signal not related to the target of interest, therefore increasing the background.
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Image-iT FX Signal Enhancer
0
100
200
300
400
500
600
700
No Block 5% NGS only Blocked Autofluorescence
Block Condition
Avg
. In
tens
ity
No Block NGS Only
Blocked with Image-IT FX Blocking Solution
Superior blocking – Boost overall signal to noise and to reduce backgroundImage-iT FX Signal Enhancer – specifically designed for use with the Alexa Fluor dyes
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Creating the Perfect Image….Image-iT™ FX Signal Enhancer – reduces non-specific binding of for
crisper images.
Without Image-iT™FX Signal Enhancer
With Image-iT™ FX Signal Enhancer
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Questions…