Multiple Immunostainings with Different Epitope Retrievals-The FOLGAS Protocol.

Area of Science:

• Biomedical research
• Histopathology
• Immunohistochemistry

Background:

• Formalin-fixed, paraffin-embedded (FFPE) tissues are crucial for diagnostics but challenging for multiplexed imaging.
• Existing multistaining protocols often face limitations with antibody compatibility and signal integrity in FFPE samples.

Purpose of the Study:

• To develop and validate a sequential multistaining protocol for FFPE specimens compatible with immunohistochemistry, immunofluorescence, and CyTOF imaging.
• To overcome limitations of traditional FFPE staining methods and enable advanced multiplexed analysis.

Main Methods:

• A novel formalin gas-phase (FOLGAS) sequential multistaining protocol was developed for FFPE tissues.
• The protocol allows for sequential antibody staining independent of primary/secondary antibody combinations and antigen retrieval methods.
• DAB-based hydrophobic masking was employed for metal-labeled primary antibodies to prevent cross-reactivity.

Main Results:

• The FOLGAS protocol successfully enabled sequential multistaining for immunohistochemistry, immunofluorescence, and CyTOF imaging.
• Histomorphologic details of the FFPE specimens were well-preserved throughout the multistaining process.
• No detectable cross-reactivity or loss of signal intensity was observed, indicating high signal fidelity.

Conclusions:

• The FOLGAS protocol offers a robust and versatile method for advanced multiplexed imaging of FFPE tissues.
• This technique expands the utility of CyTOF imaging in FFPE sections, facilitating deeper biological insights from archival samples.