FITC (fluorescein isothiocyanate) conjugated anti-horse primary antibodies are green-fluorescent immunoreagents designed for the detection of equine antigens in fluorescence-based applications. Produced by immunizing hosts such as goat, rabbit, or mouse with purified horse immunoglobulins, these antibodies are affinity-purified and covalently conjugated to FITC, a widely used fluorophore with excitation and emission maxima of approximately 495 nm and 519 nm, respectively. The isothiocyanate group reacts with primary amines on lysine residues to form stable thiourea bonds. Typical fluorophore-to-protein (F/P) ratios range from 3 to 8, providing an optimal balance between fluorescence intensity and preservation of antigen-binding activity. Owing to its compatibility with standard 488 nm argon-ion lasers and FITC filter sets, FITC remains one of the most commonly used fluorophores in immunofluorescence. Available formats include anti-IgG (H+L), anti-IgG subclass (e.g., IgG1, IgG2), and anti-IgM antibodies, with extensive cross-adsorption against mouse, rat, rabbit, and human immunoglobulins to minimize nonspecific binding.
Applications
FITC-conjugated anti-horse primary antibodies are extensively used in flow cytometry and fluorescence-activated cell sorting (FACS) for immunophenotyping equine leukocytes and detecting cell surface markers including CD4, CD8, CD11a, and MHC class II. In immunofluorescence microscopy, they enable the visualization and localization of target antigens in tissue sections and cultured cells through their characteristic bright green fluorescence. They are also employed in ELISA using anti-FITC detection systems or direct fluorescence readouts, as well as in fluorescence microscopy, confocal imaging, fluorescent Western blotting, and multiplex bead-based assays.
Performance and Experimental Considerations
Although FITC is valued for its versatility and broad compatibility with fluorescence instrumentation, it is susceptible to photobleaching and exhibits pH sensitivity, performing optimally between pH 7.0 and 9.0. These characteristics may limit its suitability for prolonged imaging experiments or applications conducted under acidic conditions. Recommended working dilutions typically range from 1:50 to 1:500 for flow cytometry, 1:100 to 1:1,000 for immunofluorescence microscopy, and 1:1,000 to 1:5,000 for ELISA, depending on the assay and antibody preparation. These reagents are intended exclusively for research use and are not approved for diagnostic or therapeutic applications.