Differential staining of collagen types in paraffin sections: A color change in degraded forms

Summary

The Herovici combination of picro methyl blue or aniline blue and picro acid fuchsin in proper proportion colors mature dense collagen red while reticulum and newly formed collagen are blue. Fetal collagen, wound healing collagen, subepidermal finely fibrillar collagen, deteriorating collagen in and over arterio-sclerotic lesions tend to take the blue stain. Arterial adventitial, deep dermal gastric submucosal collagens are red.

Denaturation of collagen with 2 N-3 N acetic acid or with neutral or slightly acid water (pH 4) at 95° C fragments coarse red collagen and turns its staining reaction toward blue. Renal and gastric mucosal reticulum and subepidermal collagen resist these treatments better than coarse collagen, the fine medial collagen of large arteries tends to retain morphologic integrity but alters its color to light blue. Clostridum histolyticum collagenase destroys the blue staining of renal, gastric mucosal and pulmonary parenchymal reticulum in 10–15 min, while it often takes an hour or more to destroy the red staining coarse collagens. Fibrous plaque collagen succumbs gradually to collagenase, while it appears to resist hot water denaturation better than the adventitial collagen of the same artery or that of deep dermis of the same patient. Unlike the allochrome and the periodic acid Schiff methods the blue Herovici reaction does not stain glomerular capillary and pulmonary capillary basement membranes.

The findings indicate the existence of tinctorially and chemically distinct collagens. The NK-19 lot of aniline blue was effective at a 1∶19 aniline blue:acid fuchsin ratio. Herovici used a 1∶2 ratio, other samples require intermediate ratios.