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Whole-slide, Quadruple Immunofluorescence Labeling of Routinely Processed Paraffin Sections

Buscone, Serena MSc*; Argentieri, Maria C. BSc; Pilla, Daniela BSc; Cattoretti, Giorgio MD*,†

Applied Immunohistochemistry & Molecular Morphology:
doi: 10.1097/PAI.0b013e31829928e7
Technical Article

Whole-slide images (WSI) have acquired a stable place in diagnostic histopathology and immunohistochemistry. Immunofluorescence (IF) techniques hold a limited and selective role in diagnostics (eg, renal and cutaneous pathology) and so far remain excluded from the digital pathology evolution, with notable exceptions, such as quantitative immunopathology. We explored the ability of a commercial fluorescent slide scanner to provide 4-color IF WSI from routinely processed tissues. With minor modifications and a careful match between filters and fluorochromes, we show that 4-color IF WSI can be obtained from routine material with negligible autofluorescence, good sensitivity, and diagnostic power.

Author Information

*Department of Pathology, Universitá degli Studi di Milano-Bicocca

Azienda Ospedaliera San Gerardo, Monza, Milano, Italy

The Aperio Scanscope FL was purchased with a grant from the Regione Lombardia (Call for Independent Research, DDG 6716 del 1/7/2009). M.C.A. was funded with generous donations by Prof. Franco Uggeri (UNIMIB) and Franco Ferrario (AO San Gerardo).

The authors declare no conflict of interest.

Reprints: Giorgio Cattoretti, MD, Department of Pathology, Universitá degli Studi di Milano-Bicocca, Via Pergolesi 33, 20900 Monza, Milano, Italy (e-mail:

Received January 24, 2013

Accepted April 26, 2013

© 2014 Lippincott Williams & Wilkins, Inc.