34 Simultaneous Fluorescence Immunophenotyping and FISH (FICTION)
457
Fig. 1. FICTION study on breast carcinoma cell line MCF-7. Combined imrnunophenotyping with monoclonal mouse anti-human estrogen receptor antibody visualized by Cy3 (red)
and FISH with the YAC I9IlIF containing the ESR gene visualized by FITC (green). One
positive cell showed strong nuclear staining (red) while the adjacent cell was negative. Both
cells showed three hybridization signals (green), indicating both ESR positive and negative
cells to contain three copies of the ESR gene
can be evaluated simultaneously. Appropriate filter sets are available from
microscope manufacturers and from filter producers. Documentation is
possible by conventional photography using a 400ASA film and exposure
times up to 20 s. Digital imaging using commercially available software
like the ISIS system supplied by MetaSystems Hard & Software GmbH
is an easy alternative.
Evaluation of FICTION analyses: For controlling the specificity of the
immunophenotyping process in FICTION, class-matched mouse antibodies, non-reactive with human tissue, should be applied on a control slide.
A major problem of evaluating FICTION slides is autofluorescence, which
must not be confused with non-specific antibody binding. Typically, autofluorescence can be observed with all different filter sets appearing dull
457
Fig. 1. FICTION study on breast carcinoma cell line MCF-7. Combined imrnunophenotyping with monoclonal mouse anti-human estrogen receptor antibody visualized by Cy3 (red)
and FISH with the YAC I9IlIF containing the ESR gene visualized by FITC (green). One
positive cell showed strong nuclear staining (red) while the adjacent cell was negative. Both
cells showed three hybridization signals (green), indicating both ESR positive and negative
cells to contain three copies of the ESR gene
can be evaluated simultaneously. Appropriate filter sets are available from
microscope manufacturers and from filter producers. Documentation is
possible by conventional photography using a 400ASA film and exposure
times up to 20 s. Digital imaging using commercially available software
like the ISIS system supplied by MetaSystems Hard & Software GmbH
is an easy alternative.
Evaluation of FICTION analyses: For controlling the specificity of the
immunophenotyping process in FICTION, class-matched mouse antibodies, non-reactive with human tissue, should be applied on a control slide.
A major problem of evaluating FICTION slides is autofluorescence, which
must not be confused with non-specific antibody binding. Typically, autofluorescence can be observed with all different filter sets appearing dull