Celsius, Richard-Allen Scientific) and taken to the research laboratory. The tissue

samples were then placed into a cassette and processed overnight in a Technicon. After

processing they were embedded in paraffin. Serial histological sections of 6 |tm were

obtained using a standard microtome and captured on glass slides from a warm water

bath. A minimum of three slides were obtained from each tissue sample, with one

stained with hematoxlyin and eosin and at least 2 processed using an

immunohistochemical technique. The slides were placed in a dry heat incubating oven

for one hour at 110 degrees Celsius to remove the paraffin medium. Then the slides were

placed and "cleared" in xylene for ten minutes to remove any residual paraffin. The

slides were then carried through descending serial alcohol reagents by placing them in

100% alcohol, then 95% alcohol and then 80% alcohol to re-hydrate. Then the slides

were washed with water. Then the slides were placed in antigen retrieval solution to

clean the samples from enzymes. The slides were then placed in the dry heat incubating

oven for thirty minutes at 110 degrees Celsius and allowed to cool for thirty minutes. All

of the slides were then three times washed for fifteen minutes each with phosphate

buffered saline (PBS) in order to remove all of the enzymatic digestion products.

Immunohistochemistry requires that target retrieval (results in an increase in staining

intensity with many primary antibodies) be performed to all formalin fixed paraffin

embedded tissue sections mounted on glass slides. The goal is to eliminate all enzymes

that may interfere with the intensity of the antibody staining.

 Immunohistochemistry

 IHC staining techniques allow for the visualization of tissue (cell) antigens. These

techniques are based on the immunoreactivity of antibodies and the chemical properties