In situ localization of two fibrillar collagens in two compact connective tissues by immunoelectron microscopy after cryotechnical processing.
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 labelling 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 microscopy. 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.
Nicolas
G
G
Centre de Microscopie Electronique, Laboratoire de Biologie Marine, CNRS UPR 9042 Roscoff, Paris, France.
Gaill
F
F
Zylberberg
L
L
D000818:Animals; D000845:Annelida / Q000737:chemistry*; D003094:Collagen / Q000032:analysis*; D003238:Connective Tissue / Q000737:chemistry*; D017110:Freeze Substitution; D005615:Freezing; D006054:Goldfish; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D011077:Polychaeta / Q000737:chemistry; D016610:Tissue Embedding; D016707:Tissue Fixation
D003094:Collagen