Pathology Research Core services include preparation of histology slides for microscopy, tissue microarray construction, immunostaining, digital image capture and analysis, review of slides, and preparation of tissue for subsequent nucleic acid or protein extraction.
The laboratory is divided into two separate areas, sample processing and imaging. The processing area includes multiple microtome cutting stations and cryostats for the sectioning of both frozen and formalin-fixed paraffin-embedded (FFPE) tissues as well as tissue microarrays. A paraffin embedding station and Histobath cryobath are available to re-embed old, archival FFPE samples and snap freeze fresh tissues.
Researchers can also have frozen samples physically dissected to enrich the percentage of benign or diseased tissue within any given block. Tissue microarrays are primarily constructed on an automated platform, but can be created manually using custom tools built by Mayo Clinic's engineering department. Automated immunostaining is provided for a variety of routine stains and customized optimization, and titration of new antibodies is available.
In the imaging area, individual tissue microarray cores and whole-tissue sections can be digitally scanned, archived and reviewed on networked computer stations within the firewall.
Frozen and paraffin sectioning
Frozen tissues are sectioned using Leica cryostats, and paraffin tissues are sectioned using Leica microtomes. These sections can be mounted on slides for hematoxylin-eosin (H and E) staining, immunohistochemistry, in situ hybridization or laser-capture microdissection. Cut cryosections can also be placed in microcentrifuge tubes for subsequent extraction of protein or nucleic acids.
External site paraffin blocks can be re-embedded in new paraffin prior to sectioning. Frozen samples that are too small and thin to be handled and sectioned without the use of mounting media can be placed in base molds and snap frozen in OCT. Fresh tissues also can be snap frozen using a Histobath cryobath, either with or without OCT per the investigator's instructions.
Heterogeneous frozen tissue blocks are enriched by physically dissecting the tissue pieces while keeping the tissues frozen. The use of pathologist-marked, H and E-stained tissues as a reference improves the percentage of either tumor or benign tissue preserved in the frozen samples.
Tissue microarray construction
Tissue microarrays are constructed on a semiautomated platform, the Alphelys MiniCore. Individual arrays made on the MiniCore system can be constructed with as many as 360 cores of 0.6-millimeter (mm) diameter or 187 cores of 1-mm diameter. Additionally, tissue microarrays with up to 60 cores of 2-mm diameter can be manually created.
Automated immunostaining is performed on Leica Bond and Dako Autostainer instruments using a variety of antibody detection chemistries, including DAB and NovaRED for use with brightfield microscopy and secondary antibodies conjugated to fluorophores for immunofluorescence microscopy.
Custom development and optimization of new antibodies
Antibodies are optimized through a variety of antigen retrieval techniques, including heat treatment with citrate buffers at various pH levels, heat treatment with ethylenediaminetetraacetic acid (EDTA) or digestion with proteases. Antibody concentrations are titrated to achieve the best signal-to-noise ratio. When possible, positive and negative control tissues and peptide competitions are included to validate the resulting staining.
Digital image capture and analysis
Whole tissue slides and tissue microarray slides are scanned, and the virtual slide can be accessed via the Mayo Clinic intranet. The images are stored on server space in a Mayo Clinic institutional server facility. Mayo manages user access to the server space and also stores, backs up and archives the resulting image data files.
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