Localization of specific mRNA’s in tissue is an important part of understanding changes identified at the transcriptomic level. Compared to traditional methods for in situ hybridization, RNAscope technology provides superior sensitivity and specificity. Chromogenic and fluorescent readout options allow for flexibility in experimental design, with ability for duplex and triplex assays, respectively. Ease of use is enabled by validated reagents and protocols, as well as available probes to over 13,000 mRNA transcripts. The CPAT core has dedicated equipment and bench space for RNAscope assays. In addition, the CPAT Core maintains a library of “commonly used” probes that are available to researchers. Investigators who wish to perform RNAscope assays enter the project online, after which they reserve time at the RNAscope bench. For users who are new to the assay, training is available. In addition, this service is valuable to SBDRC members who want to try the technology before investing in the equipment for their own labs.
Laser Capture Microdissection (LCM) enables isolation of homogeneous subsets of cells from the same tissue section, for example adjacent areas of epidermis that contain squamous cell carcinoma and normal epidermis. From the isolated cells, RNA and DNA is purified for use in downstream assays such as QPCR, next-generation-sequencing, and microarrays. Investigators first meet with CPAT Core personnel to discuss goals and experimental design, after which tissue blocks (paraffin) are supplied to the core for cutting and collection of sections on polyethylene napthalate (PEN)-membrane slides, and light counterstaining with H&E, all using RNase-free conditions. For added flexibility, investigators have access to protocols, training, and all necessary equipment to cut and stain their samples. Following training on a state-of-the-art Leica LMD 7000 microscope, investigators collect cells and isolate nucleic acids. Protocols and equipment for training are available for isolation, quantitation, and quality assessment of nucleic acids, and for preparation of libraries for exome-seq and RNA-seq analysis. Microarrays are carried out in collaboration with the PENN Genomics Analysis Core
In addition to morphology, analysis of molecular changes are critical for a mechanistic understanding of homeostatic and pathologic cutaneous biology. Analysis of protein expression and signal transduction is routinely carried out by the CPAT Core using immunohistochemistry and immunofluorescence on paraffin and frozen sections of skin. Many of the sample types processed for FFPE can also be prepared by the CPAT Core as frozen tissue and cryosectioned onto glass slides, for which protocols and training are also available. Immunostaining includes single and double-label assays for kinase activity (phophoepitopes), signaling molecules, immune cells, proliferation, differentiation, and apoptosis. Non-immune assays such as TUNEL staining for apoptotic cells is also available. Protocols have been optimized for skin in order to obtain maximal specificity and sensitivity, for example by using antigen-retrieval, blocking sources of non-specific signal, and purification and titration of primary antibodies. In addition, the core can develop customized protocols for novel antibodies.
Normal and diseased archived human skin tissue is critical for translational studies. The CPAT Core has access to an extensive and well-characterized repository of clinical samples that are formalin-fixed and embedded in paraffin wax. Following consultation with the CPAT Core Director, paraffin blocks from selected clinical cases are ordered using an online request form, and then delivered to the core lab. Following submission of an online order for microtomy, slides are generated by the core for histological stains, immunoassays, RNAscope in situ hybridization, and nucleic acid isolation from laser capture microdissectate.
Microscopic examination of tissue morphology is an important initial step in the phenotypic analysis of normal and diseased cutaneous tissue. Optimal histologic skin preparations are central to this analysis. For paraffin processing, tissue samples are routinely fixed, trimmed, placed in cassettes by the investigator. Protocols, training, and equipment are available for fixation, decalcification (if needed), trimming of tissue, and microtomy. Non-routine samples are fixed by the investigator and delivered to the core for trimming and cassette preparation, as well as relevant post-fixation treatments. Following delivery, samples are processed, embedded in paraffin wax and sectioned onto glass slides. Additional microtomy services include step and serial sectioning, as well as methods for nail and bone. In addition to automated H&E staining, a variety of special histochemical stains are available, including Fontana-Masson for melanin, Masson-Trichrome for collagen, and Luna’s stain for elastin and mast cells. In addition to standard samples of mouse, human, and pig skin, the core has expertise with a variety of other skin specimens that include xenografts, ex vivo organ culture of full thickness skin and microdissected scalp hair follicles, in vitro / in vivo organotypic constructs, reconstituted hair follicle patch/chamber constructs, tumors, and skin wounds. Frequently, gene expression is examined on other tissues which the core can also process, such as embryos, nail, teeth, tongue, oral mucosa, palate, mammary gland, and various internal organs.