Heat-induced antigen retrieval by using Tris-EDTA solution destroys nuclear structure in certain tissues

Area of Science:

• Histopathology
• Immunohistochemistry
• Cell Biology

Background:

• Tris-EDTA buffer (pH 9.0) is a common method for heat-induced antigen retrieval (HIAR) in immunohistochemistry.
• Its suitability for all tissues and antigen types (nuclear vs. non-nuclear) is not fully understood.

Purpose of the Study:

• To systematically evaluate the impact of Tris-EDTA HIAR on nuclear morphology and antigen detection in various mouse and human tissues.
• To compare Tris-EDTA HIAR with citrate buffer (pH 6.0) for detecting both nuclear and non-nuclear antigens.

Main Methods:

• Assessment of nuclear morphology using DAPI and hematoxylin staining.
• Immunohistochemistry and immunofluorescence assays to detect nuclear and non-nuclear antigens.
• Comparative analysis of Tris-EDTA and citrate buffer for antigen retrieval.

Main Results:

• Tris-EDTA HIAR caused selective nuclear disruption and content leakage in thyroid, lung, and kidney tissues, leading to poor nuclear antigen detection.
• Nuclear antigen detection was preserved using citrate buffer in these sensitive tissues.
• Tris-EDTA HIAR generally yielded stronger staining for non-nuclear antigens compared to citrate buffer.
• Nuclear morphology was influenced by both pH and retrieval method in sensitive tissues.

Conclusions:

• Tris-EDTA HIAR requires caution for nuclear antigen detection in thyroid, lung, and kidney tissues due to potential nuclear disruption.
• Tris-EDTA HIAR remains effective for detecting non-nuclear antigens.
• Findings offer guidance for optimizing antigen retrieval protocols in diagnostics and research.