Related Experiment Video
Updated: May 6, 2026
High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research
Published on: June 10, 2025
Whole-slide, quadruple immunofluorescence labeling of routinely processed paraffin sections
Serena Buscone1, Maria C Argentieri, Daniela Pilla
1*Department of Pathology, Universitá degli Studi di Milano-Bicocca †Azienda Ospedaliera San Gerardo, Monza, Milano, Italy.
Insights
This study demonstrates that 4-color immunofluorescence whole-slide images (IF WSI) can be generated from routine tissues using a commercial scanner. This digital pathology advancement offers diagnostic potential for immunofluorescence techniques.
Area of Science:
- Digital Pathology
- Histopathology
- Immunofluorescence Imaging
Background:
- Whole-slide images (WSI) are standard in diagnostic histopathology and immunohistochemistry.
- Immunofluorescence (IF) techniques are selectively used in diagnostics (e.g., renal, cutaneous pathology) and largely absent from digital pathology.
- Quantitative immunopathology is an exception to the exclusion of IF from digital pathology.
Purpose of the Study:
- To assess a commercial fluorescent slide scanner's capability for producing 4-color IF WSI from routinely processed tissues.
- To evaluate the feasibility of integrating IF into digital pathology workflows.
Main Methods:
- Utilized a commercial fluorescent slide scanner.
- Employed minor scanner modifications.
- Carefully matched filters and fluorochromes for optimal signal detection.
- Processed routinely available tissue samples.
Main Results:
- Successfully generated 4-color IF WSI from routine tissues.
- Achieved negligible autofluorescence.
- Demonstrated good sensitivity and diagnostic power of the generated IF WSI.
- Validated the use of standard materials for IF WSI production.
Conclusions:
- 4-color IF WSI can be reliably obtained from routine tissues using commercial scanners with appropriate adjustments.
- This technique holds significant potential for advancing diagnostic capabilities in digital pathology for IF applications.
- The generated IF WSI possess diagnostic power comparable to traditional methods.
Abstract:
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.
