Antibodies validated for routinely processed tissues stain frozen sections unpredictably.

Area of Science:

• Immunohistochemistry
• Tissue Processing
• Antibody Validation

Background:

• Reproducible antibody staining in tissues necessitates rigorous validation.
• Current antibody validation guidelines primarily focus on formalin-fixed, paraffin-embedded (FFPE) tissues.
• The suitability of antibodies validated for FFPE tissues on other fixation types remains largely unevaluated.

Purpose of the Study:

• To assess the performance of antibodies validated for FFPE tissue when applied to lightly fixed frozen sections.
• To determine the reliability of established antibody validation criteria across different tissue fixation methods.

Main Methods:

• A panel of 126 antibodies, previously validated for FFPE tissue using extended criteria, was tested.
• Antibody performance was evaluated on lightly fixed frozen tissue sections.
• Comparison of staining results between FFPE and frozen tissue sections was performed.

Main Results:

• Only 30% of antibodies performed consistently across both fixation methods.
• 35% of antibodies showed a preference for one fixation type over the other.
• 13% of antibodies exhibited non-specific staining, and 23% failed to stain effectively on frozen sections.

Conclusions:

• Antibody performance is highly dependent on the specific antigen fixation method.
• The paratope's interaction with the fixed antigen varies significantly with different fixation techniques.
• Established antibody panels require revalidation for new tissue fixation and processing protocols to ensure staining accuracy and reproducibility.