Immunocytochemical strategies for electron microscopy: choice or compromise

Abstract

SummaryThis review attempts to describe the major immunocytochemical strategies that are in current use for transmission electron microscopy. Emphasis is placed on suggesting the most appropriate application of each technique that is described. The review is not intended to identify the microscopist's`cure all' technique for immunocytochemistry but rather to describe what techniques are available and what are their relative merits. Expensive equipment and technically complicated methods are not always necessary to collect robust data. I hope to present a review that will enable its readers to select the most profitable technique (or compromise) available, to reflect both their resources and their ability to address their biological questions with vigour.

An Official Research Policy Statement of the American Thoracic Society/European Respiratory Society: Standards for Quantitative Assessment of Lung Structure

“…Formaldehyde is a good generalpurpose fixative for LM, diagnostic pathology, immunohistochemistry and immunocytochemistry with/without antigen retrieval techniques, because it does not completely destroy protein immunogenicity (54,55). However, formaldehyde does not adequately stabilize tissue structure; the fixed lung is subject to significant mechanical distortion and collapse.…”

“…Quantification of lung ultrastructure requires a fixation-processing protocol designed for transmission EM, which in the case of immunogold labeling must also expose epitopes, necessitating a compromise between preservation of ultrastructure and antigenicity (SECTION 3) (54,55,80). Many lung diseases are associated with hyperplasia and/or hypertrophy of certain cell types (e.g., type II alveolar epithelial cells).…”

Dynamics of Carbon-Concentrating Mechanism Induction and Protein Relocalization during the Dark-to-Light Transition in Synchronized Chlamydomonas reinhardtii

“…For immunogold labeling of Rubisco and CAH3, fixed and embedded samples were treated to remove superficial osmium and unmask epitopes (Skepper, 2000). Grids were incubated face down on droplets of 4% (w/v) metaperiodate for 15 min and then washed with distilled water.…”

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function

“…Samples were prepared by freeze substitution as described earlier (Skepper, 2000). Thin sections of HeLa cells expressing Beclin-1-GFP in the absence or presence of wt-Bcl-2, mit-Bcl-2 or ER-Bcl-2 were incubated with polyclonal antibody anti-GFP followed by incubation with gold-conjugated goat antirabbit secondary antibody (British BioCell International, Cardiff, Wales).…”