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Updated: Aug 8, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
[Antigen retrieval immunohistochemistry under the influence of pH value and time]
Juan Du1, Xue-ying Shi, Jie Zheng
1Department of Pathology, Peking University School of Basic Medical Sciences, Beijing 100083, China. dujuan.122@eyou.com
Insights
Optimizing antigen retrieval is crucial for accurate immunohistochemistry. EDTA-Tris buffer (pH 9.0) with specific times enhances staining for P504S, P63, and Ki-67, while CD10 shows broader optimal conditions.
Area of Science:
- Histopathology
- Immunohistochemistry
- Biomarker Detection
Context:
- Standardized immunohistochemical (IHC) staining protocols are essential for reliable diagnostic and research applications.
- Antigen retrieval methods significantly impact the sensitivity and specificity of antibody-epitope binding.
- Optimizing antigen retrieval conditions is critical for various biomarkers including P504S, P63, CD10, and Ki-67.
Purpose:
- To determine the optimal antigen retrieval buffer and duration for immunohistochemical staining of P504S, P63, CD10, and Ki-67.
- To compare the efficacy of different antigen retrieval buffers (CA pH 6.0, EDTA pH 8.0, EDTA-Tris pH 9.0) and retrieval times (12-30 min).
Summary:
- Immunohistochemical staining of prostate carcinoma (P504S), benign prostate hyperplasia (P63), proliferative breast disease (CD10), and human papillomavirus-infected tissue (Ki-67) was performed using EnVision's method.
- Optimal antigen retrieval for P504S was achieved with EDTA-Tris buffer (pH 9.0) for 20 min, P63 for 30 min, and Ki-67 for 25 min.
- CD10 demonstrated optimal staining with various buffers at 20-25 min, indicating differential antigen sensitivity to retrieval conditions.
Impact:
- Establishes specific, optimized antigen retrieval protocols for key diagnostic markers.
- Provides a foundation for improving the consistency and accuracy of immunohistochemical staining in pathology.
- Highlights the need for tailored antigen retrieval strategies based on the specific antibody and tissue type.
Objective:
To find the best antigen retrieval buffer and retrieval time for the immunohistochemical staining of P504S, P63, CD10, and Ki-67.
Methods:
The following tissues were fixed in 10% formalin: the tissues of prostate carcinoma, benign prostate hyperplasia, proliferative breast disease and cunnus epithelial tissue infected by human papilloma virus. Immunohistochemical stainings for these tissues were processed by EnVision's methods. The first antibodies were P504S for prostate carcinoma, P63 for benign prostate hyperplasia, CD10 for proliferative breast disease, and Ki-67 for cunnus epithelial tissue infected by human papilloma virus. The same second antibody EnVision was used for all the tissues. For each tissue, we used three different antigen retrieval buffers, CA (pH 6.0), EDTA (pH 8.0) and EDTA-Tris (pH 9.0). For each buffer, there were 4 durations for antigen retrieving, namely 12, 20, 25 and 30 min. The positive signals in different conditions were observed and compared under light microscope.
Results:
The best signal for P504S was observed after retrieval in the EDTA-Tris buffer (pH 9.0) for 20 min, P63 for 30 min, and Ki-67 for 25 min. The best positive signal for CD10 was observed after retrieval in all the buffers with different pH value for 20 or 25 min.
Conclusion:
Different antigens have their own optimal retrieval buffers and the optimal retrieval time. Relatively, for the antigen retrieval of Ki-67, P504S and P63, EDTA-Tris buffers with pH 9.0 is better than other low pH retrieval buffers.
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