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High-pressure freezing followed by cryosubstitution as a tool for preserving high-quality ultrastructure and immunoreactivity in theXenopus laevispituitary gland

Authors
Journal
Brain Research Protocols
1385-299X
Publisher
Elsevier
Publication Date
Volume
15
Issue
3
Identifiers
DOI: 10.1016/j.brainresprot.2005.07.001
Keywords
  • High-Pressure Freezing
  • Cryosubstitution
  • Immunogold Labelling
  • αMsh
  • Bdnf
  • Melanotrope Cells
Disciplines
  • Biology
  • Chemistry

Abstract

Abstract Subcellular localisation of proteins and peptides yields fundamental information about cell functioning. Immunoelectron microscopy is a powerful tool to achieve this goal, but combining good tissue preservation with strong immunoreactivity is a great challenge in electron microscopy. We have applied a novel approach, using high-pressure freezing (HPF) followed by cryosubstitution, to prepare the pituitary gland of the amphibian Xenopus laevis for immunogold-electron microscopy. In this way, we investigated the subcellular distribution of brain-derived neurotrophic factor and the amphibian neurohormone mesotocin in the pituitary neural lobe, and the peptide hormone α-melanophore-stimulating hormone and its protein precursor proopiomelanocortin in melanotrope cells of the pituitary intermediate lobe. In contrast to conventional chemical fixation (followed by cryosubstitution), HPF not only revealed strong immunoreactivity of the secretory products, but also provided excellent ultrastructural preservation of cell organelles, including secretory granule subtypes. We conclude that HPF followed by cryosubstitution provides a preparation technique of choice when both optimal tissue ultrastructure and strong immunoreactivity are required.

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