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The mouse model characterized by spontaneous lung metastasis from JygMC (A) cells closely resembles the human triple negative breast cancer (TNBC) subtype. The primary tumors morphologically present both epithelial and spindle-like cells, but metastases in lung parenchyma display only adenocarcinoma properties. In the study of molecular signatures, laser capture microdissection (LCM) on frozen tissue sections was used to separate the following regions of interest: the epithelial-mesenchymal transition (EMT), mesenchymal-epithelial transition (MET), carcinoma, lung metastases, normal mammary gland and normal lung parenchyma. NanoString was selected for the study of molecular signatures in LCM targets as a reliable downstream gene expression platform allowing analysis of tissue lysates without RNA extraction and amplification. This chapter provides detailed protocols for the collection of tissue, LCM sample preparation and dissection, production of lysates, extraction, and quality control of RNA for NanoString analysis, as well as the methodology of Nanostring gene expression profiling experiment.

Laser Capture Microdissection has earned a permanent place among modern techniques connecting histology and molecular biology. Laser Capture Microdissection has become an invaluable tool in medical research as a means for collection of specific cell populations isolated from their environment. Such genomic sample enrichment dramatically increases the sensitivity and precision of downstream molecular assays used for biomarker discovery, monitoring disease onset and progression, and in the development of personalized medicine. The diversity of research targets (cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, frozen tissues, archival repository slides, etc.) and scientific objectives present a challenge in establishing standard protocols for Laser Capture Microdissection. In the present chapter, we share our experiences in design and successful execution of numerous diverse microdissection projects, and provide considerations to be taken into account in planning a microdissection study. Our workflow and protocols are standardized for a wide range of animal and human tissues and adapted to downstream analysis platforms.

Biomarker discovery is a crucial part of the fast developing field of personalized medicine. Antibody-based techniques including immunostaining of tissue samples are widely used for biomarker evaluation in preclinical and clinical studies. When used in conjunction with robust image analysis methods, it provides a powerful means to assess biomarker modulation, toxicity, and patient response to targeted agents. Here, we describe the optimization of immunofluorescent (IF) staining protocols and a sample IF multiplex protocol suitable for colocalization image analysis.

The need for large-scale collection of rodent embryos and individual embryonic tissues for genomic and proteomic studies requires modification of traditional practices of embryo necropsy. The sample intended for transcriptome study should be rapidly dissected and stabilized to preserve its molecular integrity. The retrieval of high-quality RNA, DNA, and proteins from the target tissue is crucial for informative molecular analysis (e.g., gene profiling on microarray platform). We present a reliable method of collection and preparation of rodent embryos for genomic studies supported by detailed protocols and RNA extraction results for different stages of mouse embryonic development.

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus(XT) instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.

Histology, as a mean of tissue visualization on a cellular level, is a fundamental tool in the study of cancer. The need for simultaneous delivery of quality histological material for pathological evaluation and subsequent genomic and proteomic studies, however, requires modification of traditional practices to include rapid isolation and stabilization of target tissue to preserve molecular integrity. Informative molecular analysis depends on the integrity of target molecules (RNA, DNA, and proteins) in the tissue during and after its collection. A reliable systematic approach to routine and genomic/proteomic sample collection and preparation presented is supported by detailed protocols.