title: Dual immunofluorescence staining of proteoglycans in human temporal bones.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Retrospective study of proteoglycan codistribution in archival human temporal bone tissues.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Chicago, Chicago, Illinois 60637-1447, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Markaryan
authors.forename: Erik G
authors.initials: EG
authors.lastname: Nelson
authors.forename: Robert I
authors.initials: RI
authors.lastname: Kohut
authors.forename: Raul
authors.initials: R
authors.lastname: Hinojosa
mesh_terms: D002102:Cadaver; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology / Q000378:metabolism; D005240:Feasibility Studies; D005455:Fluorescent Antibody Technique; D006801:Humans; D007202:Indicators and Reagents; D011509:Proteoglycans / Q000276:immunology* / Q000378:metabolism; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013701:Temporal Bone / Q000276:immunology* / Q000473:pathology; D016610:Tissue Embedding
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D007202:Indicators and Reagents; D011509:Proteoglycans