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- Bentham, J (author)
A double-staining technique for detection of growth hormone and insulin-like growth factor-I binding to rat tibial epiphyseal chondrocytes.
- Article/chapterEnglish1993
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- 1993
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- LIBRIS-ID:oai:gup.ub.gu.se/91647
- https://gup.ub.gu.se/publication/91647URI
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- Language:English
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- In the present study a double-staining technique was developed to investigate simultaneous GH and insulin-like growth factor-I (IGF-I) binding to chondrocytes in a monolayer cell culture. Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in monolayer. GH and IGF-I were labelled with biotin. The affinity of the biotin-labelled ligands was compared with unlabelled ligands in a radioreceptor assay. To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation with Vectastain ABC complex, which was then reacted with diaminobenzidine (DAB). Double staining was accomplished by carrying out the first reaction with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphate to give a brown stain. The presence of type-II collagen was demonstrated by immunohistochemistry and used as a marker for differentiated chondrocytes. Biotin-labelled GH and biotin-labelled IGF-I exhibited dose-dependent displacements of 125I-labelled GH and 125I-labelled IGF-I respectively from the chondrocytes in a radioreceptor assay. The displacement curves were identical to those of unlabelled ligands indicating that the affinity was unaltered. Binding of biotinylated GH to cells was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly localized to cells at high density; areas which also showed a high degree of staining for type-II collagen.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject headings and genre
- Animals
- Biotin
- Cells
- Cultured
- Collagen
- metabolism
- Growth Hormone
- metabolism
- Growth Plate
- cytology
- metabolism
- Histocytochemistry
- Immunohistochemistry
- Insulin-Like Growth Factor I
- metabolism
- Isotope Labeling
- Male
- Neuropeptides
- metabolism
- Protein Binding
- physiology
- Radioligand Assay
- Rats
- Rats
- Sprague-Dawley
- Staining and Labeling
- methods
- Tibia
- cytology
Added entries (persons, corporate bodies, meetings, titles ...)
- Ohlsson, Claes,1965Gothenburg University,Göteborgs universitet,Institutionen för invärtesmedicin,Institute of Internal Medicine(Swepub:gu)xohlcl (author)
- Lindahl, Anders,1954Gothenburg University,Göteborgs universitet,Institutionen för laboratoriemedicin, Avdelningen för klinisk kemi/transfusionsmedicin,Institute of Laboratory Medicine, Dept of Clinical Chemistry/Transfusion Medicine(Swepub:gu)xlandy (author)
- Isaksson, Olle,1943Gothenburg University,Göteborgs universitet,Institutionen för invärtesmedicin,Institute of Internal Medicine(Swepub:gu)xisaol (author)
- Nilsson, Anders,1958Gothenburg University,Göteborgs universitet,Institutionen för de kirurgiska disciplinerna, Avdelningen för ortopedi,Institute of Surgical Sciences, Department of Orthopaedics(Swepub:gu)xniand (author)
- Göteborgs universitetInstitutionen för invärtesmedicin (creator_code:org_t)
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- In:The Journal of endocrinology137:3, s. 361-70022-0795
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