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In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody
S Veronese1, M Gambacorta, B Falini
1Istituto di Anatomia Patologica, Ospedale di Niguarda, Milan, Italy.
Insights
Detecting proliferating cells using bromodeoxyuridine (BrdU) requires specific tissue preparation. Bouin
Area of Science:
- Histopathology
- Cell Biology
- Immunohistochemistry
Background:
- Bromodeoxyuridine (BrdU) incorporation into DNA labels proliferating cells.
- Immunohistochemistry is a common method for detecting BrdU.
- Tissue fixation and DNA denaturation methods can affect BrdU detection.
Purpose of the Study:
- To investigate the impact of various fixatives and DNA denaturation techniques on anti-BrdU antibody reactivity.
- To determine optimal methods for in situ detection of BrdU-labeled cells.
Main Methods:
- Human tissues were incubated in vitro with BrdU.
- Immunohistochemical staining was performed using an anti-BrdU monoclonal antibody.
- Different fixatives (Bouin's fluid, formalin) and DNA denaturation methods (pepsin digestion, acid, thermal) were tested.
Main Results:
- Bouin's fluid fixation optimally preserved BrdU antigenicity in proliferating cells.
- Formalin fixation resulted in poor BrdU reactivity, which could be partially restored by pepsin digestion but with morphological artifact.
- Acid and thermal DNA denaturation techniques yielded comparable results.
Conclusions:
- Bouin fixation is superior to formalin for preserving BrdU antigenicity.
- Acid or thermal DNA denaturation is effective for detecting BrdU.
- The recommended method for in situ detection of S-phase cells using anti-BrdU is Bouin fixation followed by DNA denaturation.
Abstract:
Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.
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