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Core Research Facilities
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Jump To:
  • Core Vision Research at the Eye and Ear Institute
  • Gene Expression/Proteomics Module
  • Tissue Culture and Histology Module
  • Image Acquisition and Analysis Module
  • Molecular Biology Module
  • Flow Cytometry Module
  • Custom Fabrication Module
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    bullet point  Core Vision Research at the Eye and Ear Institute
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    See the Difference

    The Department of Ophthalmology at the University of Pittsburgh’s Eye and Ear Institute has long held a prestigious competitive Core Facility grant award from the National Eye Institute. This funding has supported our vision researchers with specialized expertise and equipment for over two decades, and contributes substantially to the Department’s reputation as a world class leader in vision research.

    What does the Core offer?
    The Core has four modules offering expertise and access to resources for different areas of research needs: (1) Imaging Acquisition & Analysis, (2) Tissue Culture & Histology, (3) Genomics, Proteomics & Molecular Biology, and (4) Flow Cytometry & Cell Sorting. Each module is staffed by a full-time dedicated research specialist, called the Module Supervisor, and it is overseen by a faculty-level Module Director (see reverse for details).

    Can I use the Core?
    The Core is available to all eligible Participating Investigators. A researcher qualifies as a Participating Investigator if they work at the University of Pittsburgh and are studying vision research. Others may utilize the Core's services only if they partner in a collaboration with a qualifying Participating Investigator who contributes funding, not merely consultation or advisory support.

    Who gets priority for Core time and resources?
    Since many investigators use the Core, the time that the Core personnel can devote to individual requests is limited, and specialized equipment must be shared. In general, usage is given to approved projects on a first-come-first-served basis. Among eligible users, those with R01 funding from the National Eye Institute receive priority over those without.

    How do the Module Supervisors provide support?
    All modules provide personnel training for equipment use and technical skill transfer, and all will consult on experimental design and data analyses. Certain modules will perform the benchwork itself on your behalf, time and resources permitting. Such benchwork is negotiated on a project-to-project basis.

    How do I get my project approved?
    The Module Supervisors generally plan their own work schedules, so you can communicate your needs directly to them. In order to receive the Director’s approval, you will need to provide the Supervisor with information about the vision-related grant that funds this aspect of your project, and any associated institutional protocol numbers (e.g. IACUC, IBC, DNA/RNA usage, etc.).

    What do I need to pay for?
    Labor is provided at no cost to the Participating Investigator. This includes time the Core Supervisor spends doing benchwork, evaluating data, and training personnel. Supplies, however, are often the responsibility of the Participating Investigator, and the policies for individual modules vary.

    What are my responsibilities as a Core user?
    You must agree to acknowledge your utilization of the Core by citing the core grant for vision research -P30 EY08098 - on your posters, in your talks, and in your manuscripts. Also, the individual modules each have rules for the proper use and care of Core equipment, which you will be expected to follow.
     
    Documents
  • Core Vision Research Print Out
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    bullet point  Gene Expression/Proteomics Module
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    Director: Paul R. Kinchington, PhD


  • The effect on human host cell gene expression of viral infection; while viruses are relatively simple biota their subversion of the host cell may cause complex and unexpected changes in host gene expression.

  • The effect of different growth factors on cells cultured from the corneal stroma. We have found that our microarray data accurately reflects some known effects of TGF-b1, i.e., immunosuppression, increased elaboration of extracellular matrix, and increased synthesis of cytoskeletal components. We are now examining the expression changes which control cell growth and intracellular signaling.

  • The effect of specific signal induction inhibitors on expression in corneal cell subtypes. For many genes, expression is likely controled by inputs from multiple signaling pathways. Identifying the small but consistent changes which occur when these individual pathways are inhibited will allow us to assemble a map of cellular control circuits.
    Current projects use predominantly Affymetrix GeneChip DNA microarrays. Microarray sample preparation and data analysis are performed within the Module, while samples are scanned in the Department of Pathology. An ABI 7700 instrument is used to perform real-time PCR, both for confirmation of microarray data and to assay the expression of genes not available on GeneChips.

  •  
    Contact Information
    Paul Kinchington
    Ph.D.
    412-647-6319
    kinchingtonp@upmc.edu
    EEINS-1020, 203 Lothrop Street, Pittsburgh PA 15213
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    bullet point  Tissue Culture and Histology Module
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    Director: James L. Funderburgh, PhD


    TISSUE CULTURE SERVICES

    Basic Cell Culture
    □ Thawing cells
    □ Maintaining cells
    □ Large culture scale-up
    □ Subculturing for experiments
    □ Cell counts
    □ Cell cryopreservation
    □ Cryostorage bank
    □ Custom media assembly (SHEM, antibiotic-free media, serum-free media)

    Advanced / Specialized Culture Services
    □ Stem Cells
    □ Mycoplasma screening of reagents cell lines
    □ Endotoxin screening of reagents / cell lines
    □ Organ culture (whole rabbit corneas)
    □ Wound healing assays (whole corneas, agarose strips, agarose dots)
    □ Rabbit primary ocular cell culture (endo, epi, stroma, conjunctiva, TM)
    □ Human primary ocular cell culture (endo, epi, stroma, TM, corneal stromal stem cells)
    □ Bovine primary ocular cell culture (keratocytes)
    □ Make stably transfected cell lines (via TurboFect, FuGene, L2K)
    □ Hybridoma services (culture cells and collect supernatant, concentrate antibody, purify antibody, modify with various tags)

    Virus Services
    □ Recombinant virus isolation – Kip’s team makes the vector, I do the transfection, isolate recombinants, and create a crude primary isolate stock.
    □ Crude stock amplification
    □ Three-tier virus cryostorage bank creation
    □ Virus purification
    □ Plaque assays – for titers of stocks, eye swabs, TGs, or other brain areas, growth kinetics


    HISTOLOGY SERVICES

    Paraffin Histology (FFPE)
    □ Protocol Formation – determine scientifically how long to fix, process, what wax is best, how long to stain for, appropriate dilutions of antibodies, etc.
    □ Tissue Fixation
    □ Tissue Processing
    □ Paraffin Embedding
    □ Sectioning on rotary microtome and mounting onto slides
    □ Deparaffinization
    □ Chemical Staining (H&E, acetylcholinesterase, blueing)
    □ Immunohistochemical Staining (DAB)
    □ Coverslipping

    Frozen Histology (Cryostat)
    □ Protocol Formation – determine scientifically how long to fix, infiltrate with sucrose, best cutting temperatures, how long to stain for, appropriate dilutions of antibodies, etc.
    □ Tissue Fixation (sometimes occurs after cutting, sometimes before)
    □ Sucrose Infiltration
    □ OCT-cassette Embedding
    □ Sectioning on Cryostat microtome
    □ Mounting onto slides
    □ Chemical Staining (H&E, acetylcholinesterase, blueing, DAPI)
    □ Immunohistochemical Staining (DAB, fluorescent)
    □ Coverslippping

    Monolayers
    □ Fixation
    □ Chemical Staining – gentian violet, alizarin red, fluorescein, DAPI, trypan blue
    □ Immunocytochemistry –DAB, fluorescent


    GENERAL SERVICES

    Personnel Training
    □ … in specific protocols (e.g. how best to grow HEK cells)
    □ … in the principals of certain techniques (e.g. aseptic technique)
    □ … for fundamentals of the scientific method (constructing a well-controlled experiment)

    General Laboratory
    □ Lot testing (of FBS, alternative companies from whom to source media)
    □ Pursue training – I attend seminars and hands-on courses related to projects I do for you.
    □ Attend your lab meetings to better understand your projects, and to add fresh insights.
    □ Encourage collaborations between you and other investigators.
    □ I buy some basic reagents (e.g. trypsin, PBS) for services I perform on your behalf.
    □ Maintain the equipment I use to perform your projects (e.g. incubators, water baths).

    Departmental Resource Oversight
    □ Dark Room
    □ Milli-Q Water System
    □ Cryostat refrigerated microtome


     
    Contact Information
    Katherine Davoli
    412-647-8256
    davolika@upmc.edu
    EEINS-928, 203 Lothrop Street, Pittsburgh, PA 15213
    Documents
  • Work Order Request Form
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    bullet point  Image Acquisition and Analysis Module
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    Director: Ian A. Sigal, PhD


    Purpose
    The Electronics/Molecular Imaging module is dedicated to providing hardware and software support for acquisition, analysis and storage of digital microscopic images.

    Laser Scanning Confocal Microscopy
    Laser scanning confocal microscopy addresses several of the problems encountered in traditional microscopy by controlling the depth of filed, eliminating image degradation through out-of-focus information and by allowing the collection of serial optical sections from thick specimens.

    Our confocal system, an Olympus Fluoview1000 is equipped with an Olympus IX81 inverted microscope, offers mercury lamp excitation for initial viewing and four lasers with six possible wavelengths of laser excitation for confocal acquisition. It has 10x, 20x and 60x air objectives with phase contrast available as well as 20x, 40x and 60x and 100x oil objectives.

    Live Cell Microscopy
    Live cell microscopy allows researchers to observe and quantify phenomena in living cells via technological advancements in hardware, software and laboratory techniques. This approach to microscopy requires creation of an environment hospitable to maintenance of cell life for the duration of the experiment.

    Our live cell system revolves around the Nikon Eclipse TE200-E inverted microscope with 4x, 10x and 20x air objectives and 40x and 60x (TIRF) objectives. Illumination is provided by a DG-4 xenon light source and a wide variety of excitation and emission filters are available. Peripherals include an Eppendorf microinjection system, a Roper CoolSnapHQ camera, uniblitz brightfield shutter, a heated objective, stage and chamber cover, x, y and z automated stage control and a suspension table. Acquisition and analysis are performed in MetaMorph.

    Services
    1. Provide training for the operation of a digital imaging microscope for stationary specimens; a confocal microscopy system; and a microscope system for automated real-time imaging and micromanipulation.
    2. Provide assistance with the techniques of acquiring and analyzing digital light, fluorescent, and confocal microscopic images, and time lapse, z-series and multiple stage position images using the MetaMorph automated imaging system.
    3. Serve as a resource for information on multiple analytical applications of different imaging software currently available at the core imaging facility, and advising on upgrades for future needs and applications.
    4. Diagnose and trouble-shooting problems with the imaging microscopes, micromanipulator and gel analysis systems.
    5. Integrate data from various imaging systems by designing or implementing software allowing importation of data into MetaMorph for more sophisticated analysis than that available in the software packages that accompany the instrument.

    Citation
    Please cite the Core Grant for Vision Research - EY08098 in your publications if you have used the services of this module.



     
    Contact Information
    Kira Lathrop
    412-647-3492
    kll21@pitt.edu
    EEINS-1026, 203 Lothrop Street, Pittsburgh, PA 15213
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    bullet point  Molecular Biology Module
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    Director: Paul R. Kinchington, PhD


    Molecular Biology is an essential component of virtually all biological research, particularly in light of the availability of a large volume of sequence information and the ready ability to manipulate it for examination of protein function. Molecular Biology is also a critical component for the follow up to modern genomic and proteomic applications. There has been an explosion in the variety of molecular applications and kits for performing them, and most are constantly changing and developing. As such, molecular biology applies to high throughput assays, manipulation and control of gene expression, mutational analysis of viruses, the host cell or the host animal. It is also a crucial component of most gene knockout and suppression strategies.

    The Molecular Biology Module is housed in two rooms (350 sq. ft., and 100 sq. ft.) on the 10th floor of the Eye and Ear Institute.

    Equipment
    The space includes a separate dark room for core equipment, phosphor imaging and UV gel analysis. Additional equipment required used by the module (ultra and high speed centrifuges, beta counter, etc) are located in a core instrument room, which is located approximately 30 feet from the Molecular Biology Module on the 10th floor. In general, work requests are prioritized on a first come-first served basis for participating NEI-funded investigators; but consideration is also given to small projects requiring immediate attention, investigators seeking pilot data for new vision-related grants, and vision-related research projects that are funded through other NIH institutes.

    Major equipment includes hybridization ovens, several vertical and slab electrophoresis apparatuses and the power packs to run them, gel drying apparatus, Stratagene DNA cross-linker used in hybridization studies, UV transilluminator, and digital camera system, cooling and two shaking water baths, refrigerated micro centrifuge, an autoclave, high speed and ultracentrifuges, spectrophotometer and analytical balances. BioRad GS625 Phosphorimager, BioRad EthBr/fluoresce Max Gel and chemiluminescent documentation system, BioRad I cyclker PCR machine, PC computer (networked), and Gene pulser for bacterial transfections. In addition, the module has had access to the electrophoresis equipment of Dr. Kinchington, and has access to P2 tissue culture facilities, high speed and ultra-centrifuges, Speedvac apparatus, -20 and -70 freezers and cold storage, and scintillation counters, all of which are housed in common facilities of the Ophthalmology Research Center and are available for common use on an equally-shared basis.

    Services
    Preparation and analysis of DNA and RNA
    Preparation and screening analysis of phage libraries and generation of subtractive libraries
    DNA mutagenesis and expression
    DNA:protein, and protein:protein interactions
    RNA mapping and accurate quantification
    Virus vector design and derivation
     
    Contact Information
    Paul Kinchington
    PhD
    412-647-6319
    kinchingtonp@upmc.edu
    EEINS-1014, 203 Lothrop Street, Pittsburgh, PA 15213
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    bullet point  Flow Cytometry Module
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    Director: Robert L. Hendricks, PhD


    The Morphology Module provides technical support for researchers requiring sample preparation for light, confocal, and electron microscopy. These services include fixation of tissues and cultured cells, embedding, section preparation, and staining. The module will also provide facilities and training for individual researchers who prepare and stain their own samples and the module maintains a library of commonly used antibodies and histological reagents available to all core grant users.

    Equipment
    The lab contains a Leica 2800 Frigocut-E cryostat, Microm HM505E cryostat, a fume hood, light microscope, dissecting scope, refrigerator-freezer and histological technique references. The 10th floor laboratory maintains a library of secondary antibodies labeled with fluorescent tags compatible with filter sets available in the Imaging and Image analysis Module. The Histology Core (2nd floor) provides an Olympus 4060E microscope, a Lancer Vibratome Series 1000, Reichert Jung Autocut Microtome (paraffin & plastics), a Leica-Jung Polycut E fully automated microtome for large sections, a new Leica CM30505 cryostat, a Leica 2145 Microtome, Leica SM 2000R semi automatic sliding microtome, 2 AO Spencer 860 sliding microtomes and 4 AO Spencer 820 rotary microtomes. For electron microscopy there is a JB-4, and 2 LKB ultramicrotomes and an LKB glass knife breaker. For tissue processing a Tissue Tek VIP processor and a Tissue Tek embedding processor are available. A full range of standard histological stains are available. The Core Facility also has access to major equipment such as –80º C freezers, a microwave processor for microwave fixation and antigen recovery methods and a thermal cycler for in situ polymerase chain reaction methods, located on the first floor of the Eye & Ear Institute.

    Services
    The module technician maintains equipment and reagents in the module laboratory. She works directly with individual researchers advising them on the appropriate fluors for the available microscope filters and secondary antibodies. She carries out fixation, sectioning, and staining of submitted samples. Staining can include standard histological stains, immunofluorescent, or in situ hybridization. The technician will also teach histological procedures to researchers that want to carry out procedures themselves. The technician is responsible for keeping records of services performed and meets on a daily basis with the module director. The module technician coordinates projects with the technicians of the Hybridoma and Tissue culture modules as well as the Molecular Biology Module for use and testing of reagents (such as monoclonal antibodies and labeled probes) common among the modules.
     
    Contact Information
    Nancy Zurowski
    412-647-1413
    nbz1@pitt.edu
    EEINS-1048, 203 Lothrop Street, Pittsburgh, PA 15213
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    bullet point  Custom Fabrication Module
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    Director: Matthew A. Smith, PhD

    The Custom Fabrication Module is designed to serve the various needs of scientific projects for custom designed research equipment. It provides access to the resources at the University of Pittsburgh in the School of Engineering, the Swanson Center for Product Innovation (SCPI), managed by Andy Holmes. The SCPI is a state-of-the-art network of laboratories and equipment for design, prototyping and manufacturing. This module provides dedicated machinist time from the SCPI to support these roles in furtherance of research projects of participating investigators.

    Module Services

    1. Custom Machining: The SCPI is fully outfitted with all the necessary machining equipment to execute designs from scratch or modify existing objects. This includes a host of standard manual machining equipment, as well as a full complement of computer numerical control (CNC) machine tools. The staff of the SCPI is highly trained in the operation of all of this equipment.

    2. Stereolithography (SLA): The SCPI has a machine capable of 3D printing complex designs. This is an additive manufacturing process where a plastic resin is used to build a 3D object from scratch. This is a rapid process that enables the researcher to move from a design to a prototype in a matter of hours. This is ideal for rapid prototyping and, if the plastic resin is an appropriate material, the production of final research-ready materials.

    3. 3D CAD design: The SCPI has licenses and computers dedicated to 3D computer-aided design software (CAD), including Creo Elements/Pro and Solidworks. This includes 3 workstations dedicated for visitor usage, as well as numerous workstations available for the SCPI staff. The SCPI staff is highly trained in the use of this software for design of custom hardware in three dimensions, including novel designs as well as duplications or adaptations of existing objects. The SCPI also has laser-scanning hardware for measuring arbitrary shapes or objects and importing them into CAD software. Researchers using the SCPI can take advantage of their expertise in design to assist them from the beginning of a project all the way through to completion.
     
    Contact Information
    Matthew A. Smith, PhD
    412-647-2313
    smithma@pitt.edu
    EEINS-914, 203 Lothrop Street Pittsburgh, PA 15213
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