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Updated: Jun 19, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
Combined effects of formalin fixation and tissue processing on immunorecognition
D Otali1, C R Stockard, D K Oelschlager
1Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Insights
Tissue processing steps, including xylene exposure, significantly impact antigen detection in immunohistochemistry. Antigen retrieval helps but doesn't fully restore immunorecognition, highlighting the need for careful protocol optimization.
Area of Science:
- Cell Biology
- Immunohistochemistry
- Cancer Research
Background:
- Aldehyde-based fixation is known to affect antigen detection in immunodetection.
- The systematic effects of cumulative tissue processing steps on immunodetection remain underexplored.
Purpose of the Study:
- To investigate the impact of aldehyde fixation and sequential tissue processing steps on immunohistochemical antigen detection.
- To evaluate the role of antigen retrieval in mitigating these effects.
Main Methods:
- Cultured DU145 (prostate) and SKOV3 (ovarian) cancer cell lines on slides.
- Assessed immunohistochemical detection of Ki67/MIB-1 and PCNA after varying formalin fixation times and cumulative tissue processing steps.
- Evaluated the effect of antigen retrieval (AR) as a variable.
Main Results:
- Each tissue processing step, beyond fixation, influenced immunorecognition of target antigens.
- Xylene exposure caused the most significant reduction in immunorecognition.
- Antigen retrieval (AR) restored much of the staining but not all, and provided consistent results across processing steps.
Conclusions:
- Tissue processing steps critically affect immunohistochemical staining outcomes.
- Antigen retrieval is crucial for optimizing staining, particularly for PCNA, and should be considered for all steps.
- Cellular variations in antigen packaging may influence the susceptibility to fixation and processing effects.
Abstract:
It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.
