Quantitative assessment of specificity in immunoelectron microscopy

John Milton Lucocq, Christian Gawden-Bone

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

In immunoelectron microscopy (immuno-EM) on ultrathin sections, gold particles are used for localization of molecular components of cells. These particles are countable, and quantitative methods have been established to estimate and evaluate the density and distribution of "raw" gold particle counts from a single uncontrolled labeling experiment. However, these raw counts are composed of two distinct elements: particles that are specific (specific labeling) and particles that are not (nonspecific labeling) for the target component. So far, approaches for assessment of specific labeling and for correction of raw gold particle counts to reveal specific labeling densities and distributions have not attracted much attention. Here, we discuss experimental strategies for determining specificity in immuno-EM, and we present methods for quantitative assessment of (1) the probability that an observed gold particle is specific for the target, (2) the density of specific labeling, and (3) the distribution of specific labeling over a series of compartments. These methods should be of general utility for researchers investigating the distribution of cellular components using on-section immunogold labeling.
Original languageEnglish
Pages (from-to)917-927
Number of pages11
JournalJournal of Histochemistry & Cytochemistry
Volume58
Issue number10
DOIs
Publication statusPublished - Oct 2010

Keywords

  • Animals
  • Histocytological Preparation Techniques
  • Humans
  • Microscopy, Immunoelectron
  • Probability
  • Sensitivity and Specificity
  • Staining and Labeling

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