Micke Patrick, Bjørnsen Tone, Scheidl Stefan, Strömberg Sara, Demoulin Jean-Baptiste, Ponten Fredrik, Ostman Arne, Lindahl Per, Busch Christer
Ludwig Institute for Cancer Research, Uppsala, Sweden.
J Pathol. 2004 Jan;202(1):130-8. doi: 10.1002/path.1496.
Laser microdissection and pressure catapulting has become a powerful tool to obtain homogeneous cell populations from tissue samples in nearly all fields of biomedical research. The isolated cells can be subsequently used for the analysis of proteins, DNA or RNA. However, the method requires physical access to the tissue surface and the sections therefore need to be air-dried and uncovered. The consequence is poor morphology, which severely reduces the potential of the technique, especially in non-homogeneous tissues or tissues with infiltrating immune cells. To overcome this limitation, a fluid cover medium was developed and the effects on frozen and paraffin wax-embedded tissue morphology were evaluated. The cover medium improved the morphology such that it was almost comparable to sections overlaid with glass coverslips. Moreover, the laser microdissection procedure was facilitated, since the medium allowed larger areas of tissues to be laser pressure-catapulted. Neither the isolation of proteins nor the extraction of genomic DNA was adversely affected by the use of the fluid cover medium. No significant differences in RNA quantity and integrity were detected by TaqMan real-time PCR for GAPDH, and microchip electrophoresis, between covered and uncovered tissue sections. In conclusion, this method provides considerably improved morphology for laser microdissection and pressure catapulting techniques without affecting RNA-dependent downstream applications. This not only facilitates established procedures, but will also extend the application to tissues that require superior morphological resolution.
激光显微切割和压力弹射技术已成为一种强大的工具，可在几乎所有生物医学研究领域从组织样本中获取同质细胞群体。随后，分离出的细胞可用于蛋白质、DNA或RNA分析。然而，该方法需要物理接触组织表面，因此切片需要风干且不覆盖。其结果是形态不佳，这严重降低了该技术的潜力，尤其是在非均质组织或有浸润免疫细胞的组织中。为克服这一局限性，开发了一种液体覆盖介质，并评估了其对冷冻和石蜡包埋组织形态的影响。该覆盖介质改善了形态，使其几乎与覆盖有盖玻片的切片相当。此外，激光显微切割过程也更加简便，因为该介质允许更大面积的组织进行激光压力弹射。使用液体覆盖介质对蛋白质分离或基因组DNA提取均无不利影响。通过针对甘油醛-3-磷酸脱氢酶（GAPDH）的TaqMan实时聚合酶链反应以及微芯片电泳，在覆盖和未覆盖的组织切片之间未检测到RNA数量和完整性的显著差异。总之，该方法为激光显微切割和压力弹射技术提供了显著改善的形态，且不影响依赖RNA的下游应用。这不仅简化了既定程序，还将该技术的应用扩展到需要更高形态分辨率的组织。
