A Modified Bleaching Method for Multiplex Immunofluorescence Staining of FFPE Tissue Sections.

Area of Science:

• Biotechnology
• Histology
• Immunohistochemistry

Background:

• Multiplex immunofluorescence (mIF) is crucial for biomarker profiling and analyzing biomarker co-relationships within tissue sections.
• The standard Cell DIVE mIF platform uses alkaline hydrogen peroxide for sequential fluorophore inactivation, but faces challenges like suboptimal bleaching, tissue degradation, and loss of antigenicity.
• These limitations hinder efficient and reliable mIF analysis, particularly in FFPE tissues.

Purpose of the Study:

• To develop and evaluate a modified photochemical bleaching technique to overcome the limitations of standard mIF bleaching protocols.
• To assess the efficiency of fluorophore removal, tissue integrity, and antigen immunogenicity using the modified method compared to the conventional approach.
• To determine the impact of the modified technique on overall processing time and its utility for multiple rounds of staining and imaging.

Main Methods:

• A modified photochemical bleaching method was developed, involving intense LED light exposure combined with hydrogen peroxide application.
• This technique was applied in repeated staining/bleaching cycles on breast and other tissue sections.
• Residual fluorescence signal, tissue integrity, and antigenicity were quantitatively and qualitatively assessed and compared against the conventional bleaching method.

Main Results:

• The modified photochemical bleaching effectively removed fluorescence signals from previous staining rounds, yielding consistent results across multiple cycles.
• Photochemical treatments using the modified method preserved sub-cellular structures and tissue antigenicity throughout the mIF process.
• Processing time for an 8-round mIF procedure was reduced from 36 to 30 hours, with significantly less tissue degradation compared to the conventional method.

Conclusions:

• The modified photochemical bleaching technique offers reliable fluorophore removal in mIF, significantly improving tissue integrity and antigen preservation.
• This enhanced method reduces overall processing time, making it a valuable tool for studying biomarker co-localization in various tissues.
• The technique facilitates more robust and efficient multiplex biomarker analysis, overcoming key limitations of conventional mIF protocols.