Hyytinen E, Visakorpi T, Kallioniemi A, Kallioniemi O P, Isola J J
Department of Clinical Chemistry, Tampere University Hospital, Finland.
Cytometry. 1994 Jun 1;16(2):93-9. doi: 10.1002/cyto.990160202.
Fluorescence in situ hybridization (FISH) and specific DNA probes for peri-centromeric repeat regions and unique sequence loci have made it possible to study chromosomal aberrations from interphase tumor nuclei. Large-scale retrospective studies on the prognostic value of interphase cytogenetics would become feasible if these techniques were readily applicable to nuclei from archival formalin-fixed tumor tissues. We describe here an improved technique for interphase FISH analysis of tumors that have been extensively fixed in formalin. The protocol aims at improving probe penetration and hybridization efficiency by inducing chromatin decondensation and swelling of the nuclei with a heat treatment in a 90 degrees C glycerol solution prior to hybridization. Using this cell pretreatment, FISH results on the detection of chromosome copy number aberrations and amplification of the c-erbB-2 oncogene from formalin-fixed, paraffin-embedded tissues were highly concordant with those from fresh tissues. In contrast to previously described methods, separate adjustments of denaturation or proteinase K digestion are not required for each sample. This method facilitates retrospective analyses of large series of tumors and is also useful for applying FISH to routine diagnostic purposes using formalin-fixed material.
荧光原位杂交（FISH）以及针对着丝粒周围重复区域和独特序列位点的特异性DNA探针，使得从间期肿瘤细胞核研究染色体畸变成为可能。如果这些技术能够很容易地应用于存档福尔马林固定肿瘤组织的细胞核，那么关于间期细胞遗传学预后价值的大规模回顾性研究将变得可行。我们在此描述一种改进的技术，用于对已在福尔马林中广泛固定的肿瘤进行间期FISH分析。该方案旨在通过在杂交前于90℃甘油溶液中进行热处理，诱导染色质解聚和细胞核肿胀，从而提高探针穿透力和杂交效率。使用这种细胞预处理方法，对福尔马林固定、石蜡包埋组织中染色体拷贝数畸变和c-erbB-2癌基因扩增的FISH检测结果与新鲜组织的结果高度一致。与先前描述的方法不同，不需要对每个样本分别调整变性或蛋白酶K消化。该方法有助于对大量肿瘤进行回顾性分析，也可用于使用福尔马林固定材料将FISH应用于常规诊断目的。
