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Journal of Histochemistry and Cytochemistry, Vol. 45, 119-128, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

In Situ Localization of Two Fibrillar Collagens in Two Compact Connective Tissues by Immunoelectron Microscopy After Cryotechnical Processing

G. Nicolasa, F. Gaillb, and L. Zylberbergc
a Centre de Microscopie Electronique, UPMC, Paris, France
b Laboratoire de Biologie Marine, CNRS UPR 9042 Roscoff and UPMC, Paris, France
c Laboratoire d'Anatomie Comparée, CNRS URA 1137, Université Paris 7-Denis Diderot, Paris, France

Correspondence to: G. Nicolas, Centre Interuniversitaire de Microscopie Electronique (CIME), Case 197, Université Pierre et Marie Curie, 7, quai Saint Bernard, 75252 Paris Cedex 05, France.

Two fibrillar collagens, the worm cuticular collagen and the vertebrate Type I fish scale collagen, both organized in a compact tissue, were localized by immunogold electron microscopy in resin sections after freeze-fixation and freeze-substitution. Identification of these two fibrillar collagens failed with the use of postembedding labeling after conventional electron microscopic processing. Positive labeling of the Type I collagen was observed in sections of fish scales freeze-fixed by either slam-freezing or high-pressure freezing, freeze-substituted in acetone with or without osmium tetroxide, and embedded in LR White. The worm cuticular collagen was detected in sections of cuticle that were freeze-fixed, freeze-substituted (necessarily with osmium tetroxide added to acetone), and embedded in either LR White or Epon. It was also detected in specimens pre-fixed by aldehydes before freeze-fixation. The Type I fish scale collagen appears to be more sensitive than the fibrillar cuticular collagen of worms to the procedures employed for postembedding immunoelectron microcopy. Our results have shown that freeze-fixation and freeze-substitution preserved the antigenicity of the fibrillar collagens organized in a compact three-dimensional network, whereas immunolabeling failed after conventional electron microscopic procedures. These cryostabilization techniques appear to be of value to improve the immunolocalization of collagens. (J Histochem Cytochem 45:119-128, 1997)

Key Words: Freeze-fixation, Freeze-substitution, Compact connective tissues, Immunogold electron, microscopy, Cuticular collagen, Type I collagen


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