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Updated: Aug 11, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative
V Ananthanarayanan1, M R Pins, R E Meyer
1The Department of Preventive Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. viju@northwestern.edu
Insights
Bouin's fixation is often unsuitable for prostate biopsies undergoing immunohistochemistry (IHC) and nuclear morphometry. While some biomarkers like Ki-67 work well, others like MCM-2 show negative staining, impacting diagnostic accuracy.
Area of Science:
- Pathology
- Biomarker analysis
- Histology
Background:
- Immunohistochemistry (IHC) and nuclear morphometry are crucial techniques in prostate cancer diagnosis.
- The choice of tissue fixative significantly impacts the quality of IHC staining and morphometric analysis.
- Bouin's fixative is an alternative to formalin, but its suitability for specific biomarkers needs evaluation.
Purpose of the Study:
- To assess the challenges and suitability of using Bouin's fixed prostate biopsies for immunohistochemistry (IHC) and nuclear morphometry.
- To compare the efficacy of IHC staining for various biomarkers in Bouin's fixed versus formalin-fixed tissues.
- To evaluate Feulgen staining in Bouin's fixed tissues for nuclear morphometry.
Main Methods:
- Archival prostate biopsies fixed in Bouin's solution and formalin were used.
- Immunohistochemical staining was performed for nuclear (MCM-2, p27, Ki-67), membrane (C-erb-B2), and other markers (CD34, AMACR).
- Feulgen staining was conducted on Bouin's fixed tissues to assess suitability for nuclear morphometry.
Main Results:
- MCM-2 staining was negative in Bouin's fixed tissues.
- p27 showed increased background and cytoplasmic staining, while C-erb-B2 exhibited non-specific luminal staining with Bouin's fixation.
- Feulgen staining was weak in Bouin's fixed tissues; however, Ki-67, AMACR, and CD34 performed comparably in both fixatives.
Conclusions:
- Bouin's fixed prostate biopsies may not be suitable for subsequent IHC and nuclear morphometry.
- Awareness of antibody suitability for Bouin's fixed tissues is essential for accurate diagnostic interpretation.
- Formalin fixation remains a more reliable choice for a broader range of IHC and morphometric analyses in prostate biopsies.
Aims:
To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies.
Methods:
Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry.
Results:
MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues.
Conclusions:
Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.
