Laser Capture Microdissection (LCM) and 3D Sample Tracking Protocol

Abstract This protocol demonstrates the use of Laser Capture Microdissection (LCM), a precise cell isolation technique, for acquiring single neurons from right atrial ganglionated plexus (RAGP) porcine heart tissue in sync with image acquisition to enable 3D location tracking of collected samples. Fresh frozen RAGP tissue sectioned and stained for neurons (Nissl stain) is processed through LCM for sample collection. The LCM workflow is comprised of four steps: instrument setup, slide loading and tissue inspection, microdissection and image acquisition and sample processing and storage. LCM collected samples were processed for gene expression experiments : High-throughput-qRT-PCR or Single cell RNA sequencing. Gene expression data along with 3D sample tracking was used to generate a functional map of porcine RAGP. The techniques described in this protocol can be adapted to a wide variety of sample and tissue types with minor modifications. This protocol is a part of a protocol pipeline that includes cryosectioning and staining protocols upstream and Tissue Mapper protocol, High-Throughput q-RT-PCR and RNA Seq protocols downstream.