Tissue Culture & Histology Module

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