F(ab’)2 Alexa Fluor Plus 555 secondary antibody in rat intestine.
Fluorescent dyes have revolutionized biological research, providing scientists the ability to visualize tissues, cells and organelles, largely through secondary antibody conjugations. The development of Invitrogen™ Alexa Fluor™ dyes was a significant milestone in fluorescent imaging as they provide enhanced brightness, photostability and signal-to-noise ratio compared to other fluorescent dyes.
When were Alexa Fluor Dyes developed?
The history of the Alexa Fluor dyes begins in the 1970’s. At the time, fluorescent labels such as fluorescein and rhodamine were available. However, limitations in the photostability and pH sensitivity fueled the launch of Invitrogen Texas Red™ dyes and others that offered greater resistance to photobleaching and improved stability.
In the 1990’s, a further improved set of fluorescent dyes was released – the Invitrogen Alexa Fluor dyes. Specifically, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, and Alexa Fluor 594 dyes were initially introduced. are among some of the key features that made the launch of Alexa Fluor dyes so significant and are all reasons why they are still so popular. Today, Alexa Fluor–labeled antibodies are widely recognized as the standard for brightness and sensitivity among fluorescent conjugates.
Comparison of photobleaching rates of Alexa Fluor 488 and Alexa Fluor 546 dyes and the well-known fluorescein and Cy®3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with Invitrogen™ Alexa Fluor™ 488 Phalloidin and mouse monoclonal anti–α-tubulin followed by Invitrogen™ Alexa Fluor™ 546 Goat anti-Mouse IgG (top series), or fluorescein phalloidin and the anti–α-tubulin followed by a commercially available Cy®3 goat anti–mouse IgG (bottom series). The images were acquired with bandpass filter sets appropriate for fluorescein and rhodamine.
Thermo Fisher’s Invitrogen Alexa Fluor dye portfolio
Since the launch of Alexa Fluor dyes, Thermo Fisher has continued to be a pioneer in the fluorescent dye revolution with their portfolio of Invitrogen antibodies that has expanded the use of the spectrum. Thermo Fisher now offers 18 different Alexa Fluor dyes from blue to near-infrared, providing unmatched multiplexing capabilities for researchers using fluorescent microscopy, flow cytometry and other fluorescence-based applications.
More recently, Thermo Fisher has launched Invitrogen Alexa Fluor Plus dyes. These dyes are designed to provide even brighter fluorescence, improved photostability, and higher signal-to-noise ratio compared to the original Alexa Fluor dyes. This makes Alexa Fluor Plus conjugated secondary antibodies especially useful for applications that require high-resolution imaging, such as super-resolution microscopy, and for the visualization of low-abundance targets or in precious samples.
Key Features of Invitrogen Alexa Fluor Plus Secondary Antibodies
- Designed to help detect and visualize low-abundance targets in precious samples
- Intense brightness and pH insensitivity yielding long-lasting results
- Higher signal-to-noise ratio than traditional Alexa Fluor secondary antibodies
- Lower cross-reactivity compared to traditional Alexa Fluor secondary antibodies
Comparison of Alexa Fluor Plus 647 with Alexa Fluor 647 secondary antibodies. Endogenous HSP90 in HeLa cells was labeled (red) with rabbit HSP90 alpha Polyclonal Antibody (Cat. No. PA3-013, 1:1000 dilution) and visualized using (A) Goat anti-Rabbit IgG (Heavy Chain), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor Plus 647 (Cat. No. A55055; used at 1 µg/mL) and (B) Goat anti-Rabbit IgG Alexa Fluor 647 secondary antibody (used at 1 µg/mL). Nuclei (green) were stained with SYTOX™ Green Nucleic Acid Stain (Cat. No. S7020). Images were captured using CellInsight™ CX7 LZR High Content Screening Platform (Cat. No. HCSDCX7LZRPRO) under 20X objective. Inset represents control cells with no primary (NP) antibody to assess noise. (C) Graph represents the signal-to-noise ratio of fluorescent intensities from HSP90 stained cells versus control cells with no primary antibody. Error bars represent mean SD of duplicate samples.
Thermo Fisher offers two antibody formats for both Invitrogen Alexa Fluor and Alexa Fluor Plus secondary antibodies including whole IgG antibodies and F(ab’)2 fragments. F(ab’)2 fragment antibodies lack the Fc region of whole IgG antibodies, and its smaller size allows it to more easily penetrate complex tissues, making them valuable staining tools.
Comparison of whole IgG with F(ab’)2 Alexa Fluor Plus 555 secondary antibody in human small intestine (Duodenum). (A) Cell junctions were stained using Invitrogen Cadherin-17 Monoclonal Antibody (Cat No. 60351-1-IG) and Invitrogen Alexa Fluor Plus 555 whole IgG secondary antibody. (B) Under matched conditions, cell junctions were stained using the same primary antibody against Cadherin 17 (Cat. No. 60351-1-IG) and Invitrogen F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Cat. No. A48287). Nuclei were stained with Hoechst 33342 (Cat. No. H3570). Images were captured on an Invitrogen EVOS M7000 at 4x magnification.
Innovative products and tools complementary to Alexa Fluor
In addition to its portfolio of dyes, Thermo Fisher has an extensive portfolio of imaging tools that are designed to seamlessly integrate with its fluorescent dyes. One particularly powerful tool is the Invitrogen EVOS™ Imaging System. These imaging platforms utilize high-resolution cameras and bright LED light sources that can be easily matched to the spectra of Alexa Fluor and Alexa Fluor Plus dyes. This selection guide makes dye selection even simpler when using EVOS Imaging Systems.
The recently launched Stain-It™ Cell Staining Simulator is one of Thermo Fisher’s newest innovative resources which allows researchers to visualize Alexa Fluor dye conjugates, Alexa Fluor Plus dye conjugates and other fluorescent reagents in the cell structure of interest and easily plan multiplex imaging experiments.
Beyond developing new dyes, Thermo Fisher has also utilized Alexa Fluor dyes in driving innovation in fluorescent imaging techniques. Invitrogen SuperBoost™ Tyramide Signal Amplification, for example, combines the brightness of Alexa Fluor dyes with poly-HRP mediated tyramide labeling to produce an ultra-sensitive method of detection in fluorescent applications.
What’s next for Alexa Fluor dyes?
The history of Thermo Fisher’s Alexa Fluor dye technology is a testament to the power of scientific innovation. From its origins at Molecular Probes to its integration with EVOS imaging systems, Alexa Fluor dyes have become an essential tool for fluorescent imaging. With new developments such as Alexa Fluor Plus dyes, the future of this family of dyes looks brighter than ever.
Learn more about Alexa Fluor secondary antibodies at thermofisher.com/alexafluor
References
- BioProbes 26 – Alexa Dyes, Tracers and Conjugates. (1998, May 23). BioProbes Journal of Cell Biology Applications.
- Fluorophores and Their Amine-Reactive Derivatives. (2010). Molecular Probes™ Handbook – A Guide to Fluorescent Probe and Labeling Technologies.
For Research Use Only. Not for use in diagnostic procedures.
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