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Chemical mechanisms of staining methods effects of collagen types and fixatives on 1 step trichrome staining

Chemical mechanisms of staining methods effects of collagen types and fixatives on 1 step trichrome staining

Journal of Histotechnology 7(4): 181-185

Gomori's 1-step trichrome stain eliminated vagaries of differentiation inherent in multi-step methods, but fine connective tissue fibers are sometimes poorly stained. Lowering the pH of the dye solution to 1.3 yielded uniform coloration of Carnoy-fixed collagen, but not of formalin-fixed material. Therefore, effects of fixatives (Carnoy's fluid, methacarn, unbuffered and buffered neutral formalin, traditional and modified Zenker-formol) on dye binding by connective tissues contianing type I, III, IV and V collagens [human] were investigated. Pretreatment of sections with Bouin's solution was essential for satisfactory dye binding. All collagens in Carnoy- and methacarn-fixed tissues were intensely stained. The coloration of coarse (type I) collagen fibers varied little with the mode of fixation. After fixation in unbuffered 10% formalin, reticulum fibers (type III), basement membranes and related structures (type IV and V collagen) were often indistinct. Buffered neutral formalin and Zenker-formol had a less deleterious effect on reticulum than on basement membranes. After fixation in modified Zenker-formol (ZnCl2 instead of HgCl2) all collagens were brightly stained. In 1-step trichrome stains, the red dye and the phosphomolybdic or phosphotungstic acid-triarylmethyl dye complex compete for binding sites in tissues that are irreversibly altered by fixatives other than Carnoy-type solutions. Clearly, unsatisfactory coloration of fine connective tissue is not inherent in the trichrome solution, but is due to alterations of collagens by fixatives.

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Accession: 004938364

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