Laser Capture Microdissection of Tertiary Lymphoid Structures from Formalin-Fixed Paraffin-Embedded Sections of Canine Cutaneous and Subcutaneous Sarcomas for NanoString Direct RNA Counting.

Laser capture microdissection (LCM) of formalin-fixed, paraffin-embedded sections is a way to analyze gene expression of morphologically distinct areas of tissue, as microscopically visualized with stained tissue sections. Herein, I describe a method for laser dissecting lymphoid aggregates in canine cutaneous and subcutaneous sarcomas and their adjacent sarcoma tissue to determine the differential expression of RNA as determined by NanoString nCounter technology. Canine soft tissue sarcomas (STS) are diversely derived mesenchymal neoplasms that, regardless of exact histogenesis, behave similarly and thus have been grouped together as a diagnostic entity. The risk of recurrence and/or metastasis depends on the extent of surgical excision and histologic grade. Lymphoid aggregates are described in these tumors but have not been characterized. In humans, lymphoid aggregates characterized as tertiary lymphoid structures (TLS) improve the prognosis of several tumors, including sarcomas. We sought to determine if RNA expressed by lymphoid aggregates in canine sarcomas was compatible with TLS RNA expression. This chapter describes tissue preparation, staining, laser capture microdissection, and RNA isolation in preparation for digital RNA counting.