Generally speaking, the probes conjugated to secondary antibodies mainly include enzymes (horseradish peroxidase [HRP], alkaline phosphatase [AP], or their derivatives such as APAAP and PAP), fluorophores (FITC, RRX, TR, PE, Rhodamine), and biotin. The choice of secondary antibody with a specific probe largely depends on the particular experiment. For Western Blot and ELISA, enzyme-labeled secondary antibodies are the most commonly used; in contrast, fluorescently labeled secondary antibodies are typically employed in cell or tissue labeling experiments (e.g., immunohistochemistry, immunocytochemistry, and flow cytometry). To achieve maximum amplification of the detection signal, the Biotin/Avidin detection system can be utilized.

Fluorochromes are dyes with photoluminescent properties, and there are various types of fluorescent dyes. Currently, the main fluorochromes commonly used for fluorescently labeling secondary antibodies are as follows:

1. Fluorescein Isothiocyanate (FITC)

Pure FITC appears as a yellow or orange-yellow crystalline powder and is highly soluble in water and alcoholic solvents. With a molecular weight of 389.4, FITC has a maximum absorption wavelength ranging from 490 to 495 nm and a maximum emission wavelength of 520 to 530 nm, emitting bright yellow-green fluorescence. FITC can be stored for many years in a cool, dark, and dry environment, making it the most widely used fluorochrome at present. As a small-molecule compound, multiple FITC molecules can be conjugated to a single antibody. IgM antibodies are usually labeled with small-molecule fluorochromes such as FITC, Cy3/Cy5, and Texas Red. The primary advantage of FITC-labeled secondary antibodies is that the human eye is relatively sensitive to yellow-green light, and green fluorescence in section specimens is typically less intense than red fluorescence. However, FITC has the drawback of rapid photobleaching and thus must be used in conjunction with an anti-bleaching agent.

2. Tetramethyl Rhodamine Isothiocyanate (TRITC), Rhodamine Red-X (RRX), and Texas Red (TR)

These rhodamine derivatives have conjugated groups with distinct absorption wavelengths (550 nm, 570 nm, 596 nm) and maximum emission wavelengths (570 nm, 590 nm, 620 nm). RRX is particularly useful in triple-labeling experiments using a laser confocal scanning microscope equipped with a krypton-argon laser, as it can be used in combination with Cy2 (or FITC) and Cy5. This is because RRX has an emission wavelength between those of Cy2 and Cy5, with minimal overlap with both. The krypton-argon laser provides excitation wavelengths of 488 nm, 598 nm, and 647 nm, which are ideal for exciting Cy2 (or FITC), RRX, and Cy5, respectively.

Since both FITC and PE can be excited by the 488 nm wavelength of an argon laser, for double-labeling applications in flow cytometry, using a phycoerythrin (PE)-conjugated probe is more suitable than rhodamine. TRITC, a derivative of rhodamine, is a magenta powder with good stability. It has a maximum absorption wavelength of 550 nm and a maximum emission wavelength of 620 nm, emitting orange-red fluorescence. This fluorescence contrasts sharply with the green fluorescence of FITC, making TRITC suitable for double-labeling or counterstaining. Due to its slow photobleaching rate, TRITC can also be used for single-labeling experiments.

3. Cyanine Dyes (Cy2, Cy3, Cy5)

Cy2-conjugated groups exhibit excitation at a wavelength of 492 nm and emit green visible light at 510 nm. Cy2 uses the same filter set as FITC. Compared to FITC, Cy2 is more photostable; however, mounting media containing phosphorylated phenylenediamine should be avoided, as this anti-bleaching agent reacts with Cy2, leading to weak fluorescence and signal diffusion after storage of stained sections.

Cy3 and Cy5 are brighter, more stable, and produce lower background signals than other fluorophore probes. Cy3-conjugated groups have a maximum excitation wavelength of 550 nm and a maximum emission wavelength of 570 nm. Due to the close proximity of its excitation and emission wavelengths to those of TRITC, the same filter set used for TRITC can be applied to Cy3 in fluorescence microscopy. Cy3 can be excited to 50% of its maximum intensity under an argon laser (514 nm or 528 nm) and to approximately 75% under a helium-neon laser (543 nm) or a mercury lamp (546 nm). Cy3 can be used for double-labeling with fluorescein and for multi-labeling with Cy5 in confocal microscopy experiments.

Cy5-conjugated groups have a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 670 nm. They can be excited to 98% of their fluorescence intensity under a krypton-argon laser (647 nm) and to 63% under a helium-neon laser (633 nm). Cy5 can be combined with many other fluorophores for multi-labeling experiments. However, since its maximum emission wavelength is 670 nm, Cy5 fluorescence is difficult to visualize with the naked eye and cannot be optimally excited by a mercury lamp. Confocal microscopy equipped with an appropriate excitation light source and a far-infrared detector is typically used to observe Cy5, and an anti-bleaching agent should be added to aqueous mounting media. The use of Cy5 is not recommended with traditional epifluorescence microscopes.

Note

Since observing fluorescence requires excitation light of a specific wavelength, the choice of fluorophore must be based on the wavelength range available from the laboratory’s existing equipment. Additionally, attention should be paid to the requirement for distinguishing between probe colors in multi-labeling experiments. For example, the difference between Rhodamine Red-X (RRX) and Texas Red (TR) is more distinct than that between Tetramethyl Rhodamine (TRITC) and Cy3. If higher sensitivity is required, Cy3 and Cy5 are brighter than other fluorophore probes.

XMJ (a Chinese distributor) represents LGC·Seracare·KPL, a world-renowned manufacturer of secondary antibodies and substrates. LGC·Seracare·KPL offers a wide range of fluorescently labeled secondary antibodies capable of detecting targets in species including humans, rats, mice, pigs, donkeys, sheep, goats, chickens, horses, rabbits, hamsters, dogs, cattle, and various bacterial strains. Furthermore, they provide antibodies of different types, such as IgG, IgA, IgM, as well as IgG(Fc) and IgG(ab’)? fragments. Below, we have summarized some commonly used fluorescently labeled secondary antibodies:

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If you are interested in the products of LGC·Seracare·KPL distributed by XMJ, please feel free to send email to info@xmjsci.com or visit the official website at www.emergexcelerate.com for more information.