Document Detail

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species.
MedLine Citation:
PMID:  2820908     Owner:  NLM     Status:  MEDLINE    
A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30-60 min at 80 degrees C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Successful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.
L Bastholm; M H Nielsen; L I Larsson
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Histochemistry     Volume:  87     ISSN:  0301-5564     ISO Abbreviation:  Histochemistry     Publication Date:  1987  
Date Detail:
Created Date:  1987-11-17     Completed Date:  1987-11-17     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0411300     Medline TA:  Histochemistry     Country:  GERMANY, WEST    
Other Details:
Languages:  eng     Pagination:  229-31     Citation Subset:  IM    
Department of Electron Microscopy, University Institute of Pathological Anatomy, Copenhagen, Denmark.
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MeSH Terms
Adrenocorticotropic Hormone / metabolism
Cytoplasmic Granules / ultrastructure
Growth Hormone / metabolism
Microscopy, Electron / methods
Pituitary Gland, Anterior / metabolism,  ultrastructure*
Reg. No./Substance:
50-00-0/Formaldehyde; 9002-60-2/Adrenocorticotropic Hormone; 9002-72-6/Growth Hormone

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