Whole-slide, quadruple immunofluorescence labeling of routinely processed paraffin sections

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.