Aim To establish a convenient, reliable and high- n contrast method of immunohistochemical double labeling, n which can be observed with ordinary optical microscopy, and the staining results can be preserved permanently. Method This double labeling method employed the single detection system, HRP conjugated second antibodies, and used the substrates of 3,3′-diaminobenzidine ( DAB) and Nickel-enhanced DAB re-spectively. When the first labeling was completed, HRP was to-tally inactivated by boiling for 4~5 min to prevent the cross-re-action in the second labeling. Results ( 1 ) Boiling for 5 min could completely inactivate HRP activity, whereas 5% H2 O2 treatment only reduced HRP activity, and residual enzyme activ-ity generated the cross-reaction between two labeling. Boiling for a few minutes did not damage positive DAB staining in the first labeling or tissue integrity. (2) There was a sharp contrast be- n tween DAB in brown and Nickel enhanced DAB in purple-blue, which also gives clean backgrounds;reversely applying the sub-strates will ruin the quality of double labeling. In addition, the above method allowed conventional mounting protocol using xy-lene and Permount, instead of a water-based mounting medium, thus the staining results could be permanently preserved. Con-clusion As the same HRP detection system is employed, this novel double labeling method is simple, convenient and reliable. The method would bear broad applications in basic and clinical research.%目的：探索一种简便可靠、清晰的免疫组化双重标记方法,可用普通光学显微镜观察,并能长期保存染色结果。方法免疫组化双标中,均使用辣根过氧化物酶( HRP)连接的二抗,底物分别选用二氨基联苯胺(3,3′-diaminobenzidine, DAB)和镍增强DAB。第一标DAB染色完成后切片煮沸4~5 min以灭活HRP,完全阻断交叉反应,然后进行第二标镍增强DAB染色。结果①切片煮沸5 min,可完全灭活残余的HRP酶活性,防止染色反应交叉；5%的H2 O2灭活HRP酶不完全,易产生交叉反应；短暂高温灭活HRP不损坏之前的特异性DAB染色或造成组织损伤。②第一标DAB的棕黄色与第二标镍增强DAB的紫蓝色反差大、背景低；颠倒底物使用次序将损害双标质量。此外,双标切片可采用传统的中性树胶封片,染色结果能恒久保存。结论采用同一种酶标记系统建立了简便、可靠的免疫组化双标新方法。该方法标记结果清晰、操作简便、试剂易得,有较大应用价值。
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