Synthesis of a Novel Chemical J5 brown Dye for Staining Specific Bone Histological Sections in Green Swordtail Fish (Xiphophorus hellerii)

The creation of aromatic pigments in 1859 revolutionized the dye industry, with azo dyes emerging as the most significant class due to their versatility, cost-effectiveness, and widespread applications in textiles, food coloring, and medical fields. The present study aimed to synthesize and evaluate J5 brown, a novel azo dye, for staining specific bone histological sections in green swordtail fish (Xiphophorus hellerii). The dye was synthesized using the diazotization and coupling reaction. Nuclear magnetic resonance (NMR) spectrometry, infrared spectrometry, and mass spectrometry (MS) were used to confirm the structure of the dye. Histological sections of the fish samples were prepared, and the dye was evaluated for its efficacy in staining fish bone tissues. The synthesized J5 brown resulted in a brown powder with a yield of 87% and a melting point of 162–164°C. When the dye's absorption properties were evaluated over a range of pH values (2–12), the results revealed maximum absorption at 430 nm between pH 2 and 8 and at 350 nm between pH 9 and 12. Solvent polarity also influenced the dye's absorption, with notable redshifts observed in polar solvents and water. J5 brown dye effectively stained fish bone tissues, outperforming Alizarin Red in highlighting bone structures with approximately 80% accuracy compared to traditional bone stains. The present study’s findings revealed that the novel dye offers a cost-effective, stable alternative for bone tissue staining, with potential applications in both clinical and research settings. Further studies will explore its suitability for staining other tissues and its toxicity profile.