Frozen tissues are sectioned using Leica cryostats. These sections can be mounted on slides for histology, 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. Section thickness can be controlled between 4 and 20 micrometers.
Paraffin-embedded tissues are sectioned using Leica microtomes. Sections are mounted on slides for hematoxylin-eosin (H&E) staining, immunohistochemistry or laser-capture microdissection. Paraffin sections can also be cut and placed into microcentrifuge tubes for subsequent extraction of protein or nucleic acids. Section thickness can be controlled between 4 and 20 micrometers.
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, heat treatment with 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.
Laser-capture microdissection instrument use
Study groups can use the core's Arcturus Veritas laser-capture microdissection instrument to isolate individual cells or clusters of cells from lightly fixed cryosections or paraffin sections mounted on either membrane or glass slides. These cells may be used for extraction of protein or nucleic acids.
The Veritas laser-capture microdissection instrument is capable of handling both normal brightfield and fluorescence microscopy, and images can be generated during the capture procedure for use in later documentation.
The Pathology Research Core provides training and information to researchers and study teams interested in using the Veritas system.
Tissue microarray construction
Tissue microarrays are constructed on a semiautomated platform, the MiniCore 2. Individual arrays made on the MiniCore 2 system can be constructed with as many as 360 cores of 0.6-millimeter diameter or 187 cores of 1-millimeter diameter. Additionally, tissue microarrays with up to 60 cores of 2-millimeter diameter can be manually created in the core. Clients requesting tissue microarray construction should have a pathologist mark the region(s) of the blocks to be cored and mounted in the array block.
External site paraffin blocks can be re-embedded in new paraffin prior to sectioning. Frozen samples too small and thin to be handled and sectioned properly without the use of mounting media are placed in base molds and snap frozen in OCT. Fresh tissues can also 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. By using H&E-stained tissues marked by a pathologist for reference, this procedure can greatly improve the percentage of either tumor or benign tissue in the remaining frozen samples. Frozen tissue may also be dissected for use in protein or nucleic acid extraction.
Digital image capture and analysis
Digital image analysis for tissue microarray slides begins with creating virtual microscope slides using a BLISS virtual microscopy system and computer system from Bacus Laboratories. The virtual slide can then be accessed via the Mayo Clinic intranet, and each individual tissue core can be scored for data analysis.
Images digitized using the BLISS instrument can be 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.
The Nanozoomer digital pathology instrument by Hamamatsu, a high-output scanning microscope, also is available for rapidly creating digital images from whole-tissue slides.
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