Background: It is very important that the tissue and cell structure can be seen during a microscopic examination due to the staining of the tissues. Although hematoxylin and eosin (H&E) is the common staining technique, there is a possibility that complementary dyes with preferential tissue affinity will enhance the characterization of different histological structures. A previously synthesized chloroquine derivative that is an azo dye was evaluated in terms of its histological staining potential.
Methods: A previously prepared azo dye, synthesized using a diazo coupling reaction between sulphanilic acid and chloroquine and named 1-(4-sulfonylphenylazo)-2-(7-chloro-4-[{4-(diethylamino)-1-methylbutyl}amino]quinoline), was further investigated. The compound was structurally characterized using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and electron-impact mass spectrometry (EI-MS). Paraffin-embedded rat liver and kidney slides were stained using the dye and analyzed by light microscopy in parallel with conventional hematoxylin and eosin staining.
Results: The structural features of the previously synthesized chloroquine based azo dye were confirmed by spectroscopic studies. The ¹H-NMR spectrum showed characteristic aromatic and aliphatic proton resonances consistent with the molecular structure, and the EI-MS fragmentation pattern corroborated this. Histological evaluation revealed that the azo dye exhibited a preferential affinity for erythrocytes in the central vein of the liver and the glomerular capillaries of the kidney. Under the examined conditions, the dye demonstrated preferential visualization of vascular erythrocytes, producing a staining pattern distinct from routine H&E without disrupting general tissue morphology.
Conclusion: The chloroquine based azo dye exhibited favorable histological staining properties with a preferential affinity toward erythrocytes in rat liver and kidney tissues. The results indicate that the dye may serve as a complementary histological reagent for the visualization of vessels. However, as this study provides preliminary qualitative observations, further investigations including quantitative image analysis, comprehensive toxicity evaluation, and assessment across a wider variety of pathological tissues are required before it can be considered for routine diagnostic applications.