Pathology analysis and sophisticated imaging methods have become increasingly important as the emphasis on translational research has grown. These methodologies also are critical for studies using animal models. Among the services provided by the Pathology and Imaging Core are advanced histology, light and electron microscopy, confocal microscopy, image analysis, and quantitative morphometry of tissue sections and cultured cells. It can also assist with FACScan analysis.
Samples (tissue or cell cultures) will be fixed by the investigator’s laboratory personnel. Technical staff is available to help embed samples in either paraffin, glycol methacrylate, Epon, OCT or gelatin, cut sections, and mount them on slides. Routine histochemical staining, as well as some special stains are also available to assess the morphological quality of the sections.
Some examples of stains that are available are: Hematoxylin/eosin, periodic acid Schiff’s, Trichrome stain, Bielschowsky’s stain to identify axons. If there is something special that your research requires, please contact the Core Director – Dr. Donna Peters.
Our staff can help the investigator develop antigen retrieval procedures and post-fixation labeling studies. The client will provide any primary and secondary antibodies and/or linkers (e.g. streptavidin) necessary for visualization by light, fluorescence, or transmission electron microscopy. Expertise is available for a variety of labeling procedures. Live cell staining procedures to visualize and quantify the viability of cells in cultured anterior segments and whole rabbit lens capsules is also available.
|Enzyme histochemistry||carbonic anhydrase, β-galactosidase|
|Immunocytochemistry||TUNEL(Terminal transferase-mediated biotin-dUTP nick end labeling), annexin V staining|
|Apoptosis||fluorescent antibodies, peroxidase-antiperoxidase method, avidin-biotin complex method, immunogold labeling|
|In situ hybridization||DNA or RNA detection|
The Pathology and Imaging Core can assist with qualitative and quantitative morphometry utilizing well established histologic procedures to measure ganglion cell loss, apoptosis, loss of cell contacts, changes in cell morphology or the organization of the cellular matrix. For example, laser protection of retinal ganglion cells in experimental glaucoma can be determined by localizing the positions of surviving ganglion cells on serial sections through the laser spots and performing mathematical 2-D reconstructions and 3-D density plots.
The core also has Zeiss’ AxioVision software for morphometry. This software allows the investigator to generate composite images of large objects, measure length, distance, area, circumference and angles and do spot counts either manually or automatically.
We have started a human eye cell depository. The goals of this service are to train investigators to isolate human eye cells or to provide “hard to isolate” human eye cells. To date, we have provided human trabecular meshwork cells (both normal and transformed), primary human corneal keratocytes, and primary ciliary muscle cells for core participants. We also have generated a lentiviral vector expressing telomerase that can be used to expand the lifespan of human eye cells.
For questions regarding Pathology and Imaging services, please contact:
Donna M. Peters, Ph.D.
1300 University Ave.
6590 Medical Sciences Center
Madison, WI 53706