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本文报道了对两种常用的免疫电镜双重金标方法——单面法和双面法应用于大鼠胰腺抗α淀粉酶和抗胰蛋白酶标记的比较研究结果.为使各组间具有可比性,所有超薄切片都是从同一标本块连续切下,标记过程也是同时进行.按不同的标记程序,分为以下各组:A,双面法标记抗α淀粉酶和抗胰蛋白酶.B.单面法标记。C,孵育一次抗α淀粉酶,先后孵育二次金标A蛋白(PAG), C1为7nm和20nm;C2为10nm和20nmPAG;C3为20nm和7nmPAG.D,单面法标记抗α淀粉酶和无关的抗组织蛋白酶D,并在二次标记间加入游离A蛋白.E,同C组,且在同一面二次PAG孵育间加入游离A蛋白。F,对照组。A、B组结果显示,这两种方法的敏感性基本一致.C组结果表明,单面法的交叉反应是由于第二次标记的PAG和第一抗血清游离的Fc段结合所引起。第二次标记的金颗粒直径必须明显大于第一次的金颗粒,才能显著降低第二次PAG与第一抗血清的交叉反应。D组结果显示第二抗血清与第一次的PAG的交叉反应很微弱.E组说明游离A蛋白可完全阻止交叉反应。双面法的标记因在切片的两个面进行,完全避免了交叉反应,但可能因超薄切片两个面的超微结构有所不同而形成误差。两种双标方法的选择,取决于需要标记的具体内容。
In this paper, two commonly used immunoelectron microscopy double gold standard methods - one-sided method and double-sided method applied to rat pancreatic anti-α-amylase and anti-trypsin marker comparative study results in order to make each group comparable , All the ultra-thin slices are cut continuously from the same specimen block, marking process is also carried out at the same time.According to different labeling procedures, divided into the following groups: A, double-sided method labeled anti-alpha amylase and anti-trypsin. Single-sided method mark. C, incubate once anti-alpha amylase, incubated two gold standard protein A (PAG), C1 is 7nm and 20nm; C2 is 10nm and 20nmPAG; C3 is 20nm and 7nmPAG.D, single- Irrelevant anti-cathepsin D, and add free protein A between secondary tags.E, same as group C, and add free protein A during the second PAG incubation on the same side. F, control group. The results of groups A and B showed that the sensitivities of the two methods were basically the same, and the result of group C showed that the single-sided cross-reaction was caused by the combination of the second labeled PAG and the first antisera free Fc. The second labeled gold particles must be significantly larger in diameter than the first gold particles to significantly reduce the cross-reactivity of the second PAG with the first antiserum. The results of panel D showed that the second antiserum had weak cross-reactivity with the first PAG, and group E showed that free A protein completely prevented cross-reactivity. The double-sided labeling, due to the two sides of the section, completely avoids cross-reaction, but may result in errors due to differences in the ultrastructure of the two sides of the ultra-thin section. The choice of two double-labeled methods depends on the specifics that need to be tagged.