Figure 3
Processing of lesions for pathological analysis of BRAF V600 mutation. (a) The suspicious lesion is biopsied and prepared for formalin-fixed, paraffin-embedded tissue. The tissue sample is immersed in 10% buffered formalin for 4–6 h, then immersed in a series of increasingly concentrated ethanol to extract water. The final dehydration occurs in xylene, after which the tissue is immersed in a series of increasingly concentrated molten paraffin. Finally, the sample is embedded in a block of paraffin suitable for serial sectioning. Serial sectioning produces ‘ribbons’ of tissue sections that can be directly mounted onto slides, deparaffinized, and then stained. Every other section is stained, typically with hematoxylin and eosin, to identify tumor margins while unstained alternate sections are used for immunohistochemistry (IHC) or to obtain DNA for genetic analyses. In contrast to the process for surgical specimens, fine-needle aspirates are taken and transferred directly from the syringe to a slide, where the material is smeared across its surface, air dried, and fixed. (b) IHC reveals cellular expression of the BRAF kinase in BRAF mutation-positive (dark gray) cells in situ. (c) RT-PCR uses a fluorescent-labeled target-specific probe allowing amplification of the target sequence and real-time quantification of PCR products. EtOH, ethyl alcohol.
