Examination of the Relationship between Immunostaining Intensity and Antigen Amount with an Antigen-immobilized Filter Model System and Sections
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To examine the relationship between immunostaining intensity and antigen amount, serially diluted cell lysates from the liver or skeletal muscle of rats were immobilized on nitrocellulose (NC) filters. The filters were stained with antibody against cytochrome P-450 (P-450) 1A, P-450 2B, P-450 reductase, albumin, monosialoganglioside 1 (GM1) or myosin by the indirect immunoperoxidase method under saturation conditions, and processed for image analysis. There was a linear relationship between immunostaining intensity owing to and the amount of P-450 2B, P-450 reductase or myosin immobilized on the filters. However, the relationship was nonlinear for P-450 1A, albumin and GM1. Subsequently, the relationship between immunostaining intensity in sections and section thickness was examined by microphotometry. The relationship was linear for P-450 2B or P-450 reductase, but nonlinear for P-450 1A, albumin, myosin or GM1. The results indicate that immunostaining intensity resulting from P-450 2B or P-450 reductase in sections is proportional to the antigen amount in sections, whereas that from P-450 1A, albumin, myosin or GM1 is not proportional to the amount. Therefore, accurate quantitative information on P-450 2B or P-450 reductase in section is obtained by measuring immunostaining intensity in sections, while measurement of the intensity owing to P-450 1A, albumin, myosin or GM1 in sections will give erroneous quantitative results.
- ACTA HISTOCHEMICA ET CYTOCHEMICA
ACTA HISTOCHEMICA ET CYTOCHEMICA 30(5), 551-556, 1997-10-01
JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY