Chemical pretreatment of growth plate cartilage increases immunofluorescence sensitivity

Molly J. Ahrens, Andrew T. Dudley

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Immunofluorescence detection of proteins in growth plate cartilage is often unsuccessful because of innate autofluorescence, fixative-induced fluorescence, and dense cartilage matrix, which can inhibit antibody penetration. To overcome these limitations, the authors have tested various chemical pretreatments, including the autofluorescence quencher sodium borohydride, the antigen retrieval method of boiling sodium citrate, sugar-degrading enzymes (hyaluronidase, heparinase, and chondroitinase), and the proteolytic enzyme protease XXIV. Here the authors show that, in most cases, background fluorescence in cartilage is the primary obstacle to high-quality imaging. Blocking intrinsic fluorescence of the specimen in combination with specific pretreatments allows visualization using antibodies that previously did not generate a robust signal in the growth plate. Each antibody requires a specific combination of chemical pretreatments that must be empirically determined to achieve optimal staining levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Original languageEnglish (US)
Pages (from-to)408-418
Number of pages11
JournalJournal of Histochemistry and Cytochemistry
Volume59
Issue number4
DOIs
StatePublished - Aug 22 2011

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Keywords

  • Antibody
  • Antigen retrieval
  • Autofluorescence
  • Cartilage
  • Immunofluorescence
  • Tissue pretreatment

ASJC Scopus subject areas

  • Anatomy
  • Histology

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