Fluorescent signals are extremely important for labeling, identifying, quantifying and localizing cellular components in tissue sections and cell culture, etc. Fluorochromes are colored dyes that accept light energy at a certain wavelength (excitation) and re-emit it at a higher wavelength (emission). Emission occurs extremely rapidly after excitation and is known as fluorescence. This occurs as a result of electrons moving between higher and lower energy states. The reduction in energy between excitation and emission results in an increase in the wavelength of light since the wavelength of light is inversely proportional to its energy. By employing a series of filters, and depending on the arrangement of lasers fitted, modern microscopes can detect the light emissions from several different types of fluorochromes, allowing the localization of up to 5 components at the same time.
Alexa® Dyes from Invitrogen (Molecular Probes) are the leader in the field. These dyes are high quality, exceptionally bright, photostable, fluorescent labels for antibodies or other proteins. In addition, Alexa® dyes are insensitive to pH in the 4–10 range. Hydrazide forms of the Alexa® dyes are very bright, formaldehyde-fixable polar tracers.
Alexa® 350, Alexa® 430, Alexa® 488, Alexa® 532, Alexa® 546, Alexa® 568, and Alexa® 594 dyes belong to a new series of fluorescent dyes with emission/excitation spectra similar to those of AMCA, Lucifer Yellow, fluorescein, rhodamine 6G, tetramethylrhodamine or Cy3, lissamine rhodamine B, and Texas Red, respectively (the numbers in the Alexa® names indicate the approximate excitation wavelength maximum in nm). Though the spectra are similar, Alexa® fluorochromes are several-fold brighter and more photostable than their spectral analogues listed above. Our evaluations show that conjugates of Alexa® 546 are at least 2-fold more fluorescent than Cy3 conjugates. We almost always use the brighter and more photostable alternative to FITC, developed by Molecular Probes, called Alexa®488; it has similar excitation (494 nm) and emission (519 nm) maxima to FITC. We find that this dye is about 5 to 6 times brighter than FITC. We also extensively use Alexa® 568 and Alexa® 594 which are 4 to 6 times brighter than the rhodamine they replace in staining protocols. Conjugates of the Alexa® 430, Alexa® 532, Alexa® 555, Alexa® 633, Alexa® 647, Alexa® 660, Alexa® 680, Alexa® 700, Alexa® 750 fluorochromes are spectrally unique fluorescent probes, with relatively high quantum yields in their excitation and emission wavelength ranges. These fluorescent conjugates are produced by Invitrogen (Molecular Probes) in a variety of species specific anti-IgG or IgM antibodies, making it very easy to perform multiple wavelength labeling (assuming the different proteins are recognized by the primary antibodies). All these products are certified to be free of unconjugated dye and are tested in cytological experiments to ensure low non-specific labeling.
We stain Drosophila embryos in early division cycles for different proteins; some of the proteins we label include tubulin for labeling microtubules, actin to localize microfilaments, aurora A kinase, KLP61F, KLP3A, peanut, diaphanous, septin 2, and anillin. After incubation for an hour with a specific primary antibody, we usually use the Alexa® dyes at a 1:50-1:100 dilution and add about 100 ml of dye diluted in PBS plus 1% Tween 20 onto each coverslip. We then incubate for about an hour in a moist chamber. We usually do triple labeling experiments using Alexa® 488, Alexa® 568 and TOTO–3 iodide also from Invitrogen (Molecular Probes). We use TOTO-3 iodide probes at a 1:10000 dilution for 10 minutes, usually to stain for DNA.
Alexa® dyes are more photostable (i.e. resistant to photobleaching) than others on the market. To further reduce photobleaching, however, it is advisable to minimize the exposure of fluorescently-labeled specimens to light with neutral density filters. Also, limit exposure only to the time when the image is acquired, use high numerical aperture objectives and low magnification. We also use antifading agents, such as paraphenylendiamine (in Tris–glycerol), in our mounting media. We use this antifade reagent because it is cheaper to make than it is to buy the ready made SlowFade® Gold or ProLong® Gold from Invitrogen (Molecular Probes).
We capture our images with a Nikon Eclipse TE2000-E inverted microscope (Nikon Mississauga, ON, Canada) equipped with a Perkin Elmer Life Sciences UltraVIEW confocal spinning disk (Perkin Elmer Life Sciences, Missisauga, ON, Canada) and a Hamamatsu Orca-ER camera (Hamamatsu Photonics, Hamamatsu, Japan).
Once Alexa® fluorochrome conjugates are diluted, they should be protected from light in storage and during staining. We usually keep them at 4ºC in aluminum jars. As we use them on a daily basis, we do not aliquot them and store them at a lower temperature. Good care must be taken not to reuse an aliquot too many times as this can lead to a loss of fluorescence over time due to repeated exposure to light.
Dr. Rosalind Silverman
University of Toronto
Department of Medical Genetics and Microbiology
Dr. Lorelei Silverman
University of Toronto
Department of Physiology