抗原修复液pH值及修复时间对免疫组化染色效果的影响

Antigen retrieval immunohistochemistry under the influence of pH value and time

目的:探讨使P504S、P63、CD10、Ki 67抗原抗体复合物在免疫组化检测中获得高表达的抗原修复条件。方法:取前列腺癌、良性前列腺增生、乳腺增生组织和人乳头状瘤病毒感染的外阴上皮组织,用 10% (体积分数 )中性缓冲甲醛固定液固定。用EnVision二步法行免疫组化染色, P504S、P63、CD10、Ki 67分别作为前列腺癌、良性前列腺增生、乳腺增生组织和感染人乳头状瘤病毒的外阴上皮组织的一抗,使用统一的EnVisionTM二抗。每种组织皆分别使用 3种不同的缓冲液进行抗原修复:pH6. 0的枸橼酸缓冲液、pH 8. 0的乙二氨四乙酸二钠 (EDTA)缓冲液和pH9. 0的EDTA Tris缓冲液。对每一种抗原修复液,应用 4个不同的抗原修复时间: 12min、20min、25min和30min。染色完成后,镜下对比不同条件下抗原抗体复合物表达的情况。结果:P504S在pH9. 0的EDTA Tris缓冲液中修复 20min阳性信号最强,阳性细胞比率最高;P63在pH9. 0的EDTA Tris缓冲液中修复 30min阳性信号最强,阳性细胞比率最高; Ki 67在pH 9. 0的EDTA Tris缓冲液中修复 25min阳性信号最强,阳性细胞比率最高;CD10在不同pH值的缓冲液中修复 20min或 25min效果相同,都能得到最强表达。结论:不同的抗原有其各自最适合的抗原修复液、pH值范围和最适修复时间。相对来说,

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 TM 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.