INSERM, U1015, Villejuif, France.
Autophagy. 2012 Aug;8(8):1175-84. doi: 10.4161/auto.20353. Epub 2012 May 31.
Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.
自噬是一种进化上保守的分解代谢过程，涉及将细胞质成分捕获在特征性囊泡内，以便将其递送至溶酶体并在其中降解。在包括癌症在内的几种人类疾病中发现了自噬信号的改变。在这里，我们描述了一种经过验证的免疫组织化学检测人福尔马林固定、石蜡包埋的癌症标本中 LC3 斑点的方案，该方案也可应用于小鼠组织。在对全身化疗的反应中，自噬功能正常的小鼠肿瘤表现出 LC3 斑点，而在 Atg5 或 Atg7 敲低导致自噬缺陷的小鼠癌症中则没有出现 LC3 斑点。与正常组织相比，LC3 染色在绝大多数研究的人类癌症中中度至高度升高，尽管同一组织学类型的肿瘤在每个细胞的 LC3 斑点数量和强度上往往高度不均一。此外，肿瘤浸润免疫细胞通常对 LC3 呈高度阳性。总之，这种 LC3 染色方案似乎适用于包括组织微阵列在内的人类标本中 LC3 斑点的特异性检测。我们推测，该技术可用于涉及大量人类肿瘤样本的回顾性或前瞻性研究。
