title: Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei.
abstract: BACKGROUND
  Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens.
  
  
  METHODS
  Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data.
  
  
  RESULTS
  The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample.
  
  
  CONCLUSIONS
  The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.
authors.affiliation: Centre for Image Analysis at Uppsala University, Uppsala, Sweden. carolina@cb.uu.se
authors.forename: Carolina
authors.initials: C
authors.lastname: Wählby
authors.forename: Fredrik
authors.initials: F
authors.lastname: Erlandsson
authors.forename: Ewert
authors.initials: E
authors.lastname: Bengtsson
authors.forename: Anders
authors.initials: A
authors.lastname: Zetterberg
mesh_terms: D000906:Antibodies / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology; D002467:Cell Nucleus; D005260:Female; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000276:immunology / Q000473:pathology; D011489:Protein Denaturation; D013194:Staining and Labeling / Q000379:methods; D002583:Uterine Cervical Neoplasms / Q000276:immunology / Q000473:pathology
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D005456:Fluorescent Dyes