A customized context-dependent validation of antibodies for the prospective use, rather than a general stamp of validity, is required for reproducibility and data validity, besides the need of standardized reagents. In-situ antibody staining is a technique broadly used in experimental settings and human diagnostic practice. The first typically, but not exclusively uses lightly fixed material (cell smears, frozen sections), the second, routinely processed formalin-fixed, paraffin-embedded (FFPE) tissue. Differently from techniques based on tissue extraction, there is little awareness of the context-dependent constraints inherent with either type of in situ staining except that antigen masking associated with routine tissue processing limits the range of useful antibodies. We applied a panel of 126 antibodies validated for FFPE to lightly fixed (acetone, formalin) frozen sections and found that less than 30% performed conservatively with all fixations, 35% preferred one fixation over another, 13% gave non-specific staining, 23% did not stain at all. Individual antibody variegation of the paratope fitness for the differentially fixed antigen may be the cause. Re-validation of established antibody panels is required when applied to sections whose fixation and processing is different from the tissue where they have been initially validated.

中文翻译：

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并非所有抗体均能达到均等的效果：经过常规处理的组织验证的抗体很少应用于冷冻的组织切片

除需要标准化试剂外，对于可重复性和数据有效性，还需要针对预期用途的定制上下文相关抗体验证，而不是一般的有效性验证。原位抗体染色是一种广泛用于实验环境和人类诊断实践的技术。第一种通常，但不是唯一地使用轻度固定的材料（细胞涂片，冷冻切片），第二种是常规处理的福尔马林固定，石蜡包埋（FFPE）组织。与基于组织提取的技术不同，除了与常规组织处理相关的抗原掩蔽限制了有用抗体的范围外，几乎没有人意识到任一种原位染色所固有的上下文相关限制。我们将经过FFPE验证的126种抗体应用于轻度固定的（丙酮，福尔马林）冰冻切片，发现在所有固定中，保守的执行率不到30％，35％的优先选择另一个固定，13％的非特异性染色23 ％根本没有染色。差异互补的抗原的互补位适应性的个体抗体变异可能是原因。将固定的抗体面板应用于固定和处理不同于最初验证的组织的切片时，需要重新验证。差异互补的抗原的互补位适应性的个体抗体变异可能是原因。当将固定的抗体面板应用于其固定和处理方式与最初经过验证的组织不同的部分时，需要对其进行重新验证。差异互补的抗原的互补位适应性的个体抗体变异可能是原因。当将固定的抗体面板应用于其固定和处理方式与最初经过验证的组织不同的部分时，需要对其进行重新验证。