Immunological probes for electron microscopy: Evaluation of staining by fluorescence microscopy

R. B. Alexander, W. B. Isaacs, E. R. Barrack

Research output: Contribution to journalArticlepeer-review

Abstract

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary and anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

Original languageEnglish (US)
Pages (from-to)995-1000
Number of pages6
JournalJournal of Histochemistry and Cytochemistry
Volume33
Issue number10
DOIs
StatePublished - Jan 1 1985

ASJC Scopus subject areas

  • Anatomy
  • Histology

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