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Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

Maddalena Maria Bolognesi1, Marco Manzoni1, Carla Rossana Scalia1

  • 1Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (MMB, MM, CRS, SZ, FMB, GC).

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|July 11, 2017
PubMed
Summary

Multiplexing allows multiple in situ immunostains on single tissue sections using common antibodies and scanners. This high-throughput method enables detailed cell characterization with modest investment.

Keywords:
antibody removalepitopeimmunofluorescencemultiplex

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Area of Science:

  • Biomedical science
  • Histopathology
  • Immunofluorescence techniques

Background:

  • Multiplexing for in situ immunostaining is of significant interest.
  • Existing methods face challenges like high costs, low throughput, and specialized skill requirements.

Purpose of the Study:

  • To validate a cost-effective, high-throughput multiplexing method for routine tissue sections.
  • To enable detailed in situ cell characterization using common reagents and equipment.

Main Methods:

  • Sequential rounds of four-color indirect immunofluorescence staining, image acquisition, and antibody stripping.
  • Antibody removal via disulfide cleavage/detergent or chaotropic salt treatment with antigen refolding.
  • Digital image registration and autofluorescence subtraction.

Main Results:

  • Successfully applied over 30 different antibody stains to a single tissue section.
  • The method is compatible with routinely fixed and embedded tissues.
  • Achieved high throughput for in situ cell characterization.

Conclusions:

  • Validated a multiplexing technique using common antibodies and widely available fluorescent scanners.
  • This method offers a high-throughput solution for in situ cell analysis with minimal hardware investment.
  • Enables comprehensive characterization of various cell types in tissue sections.