Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.
Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry.
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DOI：
10.4081/ejh.2015.2481
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发表时间：
2015-05-05
期刊：
影响因子：
--
通讯作者：
Tschan MP
中科院分区：
文献类型：
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作者：
Schläfli AM;Berezowska S;Adams O;Langer R;Tschan MP
自噬可确保细胞稳态，并在癌症中变得越来越重要，它影响癌发生、恶性表型的传播和耐药性的发展。迄今为止，通过免疫组织化学进行的基于组织的分析仍然缺乏标准化。在这里，我们展示了通过免疫组织化学特异性、可靠地评估福尔马林固定和石蜡包埋的人体组织中自噬标记物 LC3B 和 p62 (SQSTM1) 的可行性。先前的功能实验包括消耗 H1299 肺癌细胞中的 LC3B 和 p62，随后诱导自噬。在福尔马林固定和石蜡包埋之前，通过蛋白质印迹和免疫荧光验证抗体特异性、敲低效率和自噬诱导。 LC3B 和 p62 抗体在处理细胞和对照细胞的福尔马林固定和石蜡包埋的细胞沉淀上进行了验证，并最终应用于包含 80 个人类恶性和非肿瘤性肺和胃福尔马林固定和石蜡包埋组织样本的组织微阵列。分别在细胞沉淀和18/40 (LC3B) 和22/40 (p62) 肿瘤中观察到不同程度的点状染色。 17 个肿瘤的 LC3B 和 p62 双阳性。 P62 显示出额外的显着细胞质和细胞核染色，但意义未知。染色强度和图案分级的观察者间协议为显着至极好（kappa 值 0.60-0.83）。总之，我们提出了对福尔马林固定和石蜡包埋的人体组织进行 LC3B 和 p62 特异性且可靠的 IHC 染色。我们提出的方案旨在帮助对实体瘤中失调的自噬进行可靠的研究，并可用于大型组织集体。
Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.