Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections.
Microsc Res Tech
; 67(1): 15-21, 2005 May.
Article
em En
| MEDLINE
| ID: mdl-16025486
ABSTRACT
Immune-cell infiltration is frequently seen within human solid tumors. A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response. Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section. Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section. This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors. We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity. Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap. For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images. This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.
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Base de dados:
MEDLINE
Assunto principal:
Imuno-Histoquímica
/
Neoplasias Colorretais
/
Biomarcadores Tumorais
/
Imunofenotipagem
/
Leucócitos
/
Microscopia
Limite:
Humans
Idioma:
En
Revista:
Microsc Res Tech
Assunto da revista:
DIAGNOSTICO POR IMAGEM
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Holanda