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.

This is a preview of subscription content, access via your institution.

References

  1. Bielschowsky, M.: Die Silberimprägnation der Neurofibrillen. J. Psychol. Neurol. 3, 169–189 (1904)

    Google Scholar 

  2. Böck, P.: Histochemieal demonstration of type IV collagen in the renal glomerulus. Histochemistry 55, 269–270 (1978a)

    Google Scholar 

  3. Böck, P.: The distribution of disulfide groups in Descemet's membrane, lens capsule and zonular fibers. Acta Histochem. 63, 127–136 (1978b)

    Google Scholar 

  4. Castino, F., Bussolati, G.: Thiosulphation for the histochemieal demonstration of protein-bound sulphydryl and disulphide groups. Histochemistry 39, 93–96 (1974)

    Google Scholar 

  5. Clark, G.: Van Gieson trypan blue. In: Staining procedures, 3 ed., p. 43 Baltimore: Williams and Wilkins 1973

    Google Scholar 

  6. Curtis, F.: Nos méthodes de coloration élective du tissu conjonctif. Arch. méd. expér. 17, 603–636 (1905)

    Google Scholar 

  7. Gustafson, K.H.: The chemistry and reactivity of collagen. New York: Academic Press, 1956

    Google Scholar 

  8. Herovici, C.: A polychrome stain for differentiating precollagen from collagen. Stain Technol. 38, 304–305 (1963 a)

    Google Scholar 

  9. Herovici, C.: Étude de la réaction du tissu conjonctif dans les tumeurs malignes du col uterin. Bull. Cancer 1, 50:519–520 (1963 b)

    Google Scholar 

  10. Herovici, C.: Étude de la réaction du tissu conjonctif dans les tumeurs malignes du col uterin. Neoplasma 11, 225–240 (1964)

    Google Scholar 

  11. Ladeweicig: Über eine einfache und vielseitige Bindegewebsfärbung Z. wiss. Mikrosk. 55, 215–217 (1938)

    Google Scholar 

  12. Lillie, R.D.: Studies on selective staining of collagen with acid anilin dyes. J. Tech. Meth. 25, 1–47 (1945)

    Google Scholar 

  13. Lillie, R.D., Laskey, A., Greco, J., Burtner, H.J.: Reticulum staining with Schiff reagent after oxidation by acidified sodium periodate. J. Lab. Clin. Med. 32, 910–912 (1947)

    Google Scholar 

  14. Lillie, R.D.: The allochrome procedure: a differential method segregating the connective tissues, collagen, reticulum and basement membranes into two groups. Am. J. Clin. Pathol. 21, 484–488 (1951)

    Google Scholar 

  15. Lillie, R.D.: Histochemistry of connective tissues: collagen, reticulum, basement membranes, sarcolemma, ocular membranes. Lab. Invest. 1, 30–45 (1952)

    Google Scholar 

  16. Lillie, R.D. and Fullmer, H.M.: Histopathologic technic and practical histochemistry 4th ed. New York: McGraw-Hill 1976

    Google Scholar 

  17. Lillie, R.D., Reynolds, C., Pizzolato, P.: Phosphomolybdic acid and phosphotungstic acid Vitoria Blue R stains in relation to dense and areolar collagen staining. J. Histochem Cytochem. 27, 1092–1094 (1979)

    Google Scholar 

  18. McManus, J.F.A.: The periodic acid routine applied to the kidney. Am. J. Pathol. 23, 507–508 (1947)

    Google Scholar 

  19. Mall, F.: Das reticulirte Gewebe und seine Beziehungen zu den Bindegewebsfibrillen. Abh. math. phy. Class. Kgl. Sachs. Gesell. Wiss. 17, 295 (1891)

    Google Scholar 

  20. Mall, F.P.: Reticulated tissue and its relation to the connective tissue fibrils. Johns Hopkins Hosp. Rep. 1, 171–206 (1896)

    Google Scholar 

  21. Maresch, R.: Über Gitterfasern der Leber und Verwendbarkeit der Methode Bielschowskys zur Darstellung feinstar Bindegewebsfibrillen. Zbl. allg. Path. path. Anat. 16, 817–819 (1905)

    Google Scholar 

  22. Sweat, F., Puchtler, H., Woo, P.: Lightfast modification of Lillie's allochrome stain. Arch. Path. 78, 73–75 (1964)

    Google Scholar 

  23. Unna, P.G.: Neue Untersuchungen über Kollagenfärbung. Monatsh. prakt. Dermatol. 24, 359–400 (1903)

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Dr. R. E. Tracy.

Additional information

Aided by NIH Grant HL 08974 National Institutes of Health

Prof. Dr. R.D. Lillie died on October 5, 1979

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lillie, R.D., Tracy, R.E., Pizzolato, P. et al. Differential staining of collagen types in paraffin sections: A color change in degraded forms. Virchows Arch. A Path. Anat. and Histol. 386, 153–159 (1980). https://doi.org/10.1007/BF00427227

Download citation

Key words

  • Arteriosclerosis
  • Fibrous plague
  • Aging
  • Wound healing