This work was supported by the Wake Forest University School of Medicine, Department of Otolaryngology, Temporal Bone Histopathology Laboratory. The authors have no other funding, financial relationships, or conflicts of interest to disclose.
Dual immunofluorescence staining of proteoglycans in human temporal bones†
Article first published online: 6 JUN 2011
Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.
Volume 121, Issue 7, pages 1525–1531, July 2011
How to Cite
Markaryan, A., Nelson, E. G., Kohut, R. I. and Hinojosa, R. (2011), Dual immunofluorescence staining of proteoglycans in human temporal bones. The Laryngoscope, 121: 1525–1531. doi: 10.1002/lary.21772
- Issue published online: 16 JUN 2011
- Article first published online: 6 JUN 2011
- Manuscript Accepted: 5 JAN 2011
- Manuscript Revised: 26 DEC 2010
- Manuscript Received: 31 OCT 2010
- Chondroitin sulfate and keratan sulfate proteoglycans;
- immunofluorescence staining;
- human temporal bones;
- Level of Evidence: 3b.
Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section.
Retrospective study of proteoglycan codistribution in archival human temporal bone tissues.
The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics.
The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis.
The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes.