title: Chemical pretreatment of growth plate cartilage increases immunofluorescence sensitivity.
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.
authors.affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Molly J
authors.initials: MJ
authors.lastname: Ahrens
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Dudley
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001894:Borohydrides; D002810:Chondroitinases and Chondroitin Lyases; D002951:Citrates; D005455:Fluorescent Antibody Technique; D006132:Growth Plate / Q000378:metabolism*; D019763:Heparin Lyase; D016591:Histocytological Preparation Techniques / Q000379:methods; D006821:Hyaluronoglucosaminidase; D051379:Mice; D012680:Sensitivity and Specificity; D000077559:Sodium Citrate; D020860:Subtilisin
chemical_list: D001894:Borohydrides; D002951:Citrates; D000077559:Sodium Citrate; C025364:sodium borohydride; D006821:Hyaluronoglucosaminidase; C430466:protease XXIV; D020860:Subtilisin; D002810:Chondroitinases and Chondroitin Lyases; D019763:Heparin Lyase