Description
Histology processing and diagnosis, as well as related techniques such as histochemistry, immunohistochemistry, autoradiography, in situ hybridization, etc. have become indispensable tools for the study of cellular responses to different environmental stimuli. In addition, the more recent use of microdissected paraffin sections in molecular biology studies (detection of mutation, polymorphisms, sequencing, etc.) have made these techniques an essential component of many research projects.
Many current and proposed research projects of CRED members utilize animal models in which the end point is the histopathological assessment of tissue pathology (e.g., toxicity, cell proliferation, neoplasia). In addition, increasing numbers of research projects utilize human tissues that must be analyzed at the microscopic level. This trend is expected to accelerate as Center Members take advantage of the resources provided by the new Integrative Health Sciences Facility Core. Many projects also generate and use transgenic mouse models that require extensive characterization of tissue phenotypes. This extensive characterization and analysis of models is evident by the increased numbers of immunohistochemical stains that have been added each year by the Core. Prior to year six, before the addition of an Immunohistochemist, the Core provided about 20 routine antibody stains. To date, the Core has added about 40 new antibodies per year for a total of about 180 antibodies. A recent addition has been the characterization of human tumor cells and tissues that are being grown in or implanted into experimental mice. Careful attention to primary and secondary antibody specificity and cross reactivity in these mixed tissues (i.e. human tumor cells with mouse vascularization) requires a high rate of technical expertise that this Core provides. The MB Core can provide quantitative examination of proteins, gene analysis, etc. that is beyond the sensitivity of Immunohistochemistry. By combining the information that the two Cores provide, CRED researchers get molecular, immunohistochemical and histopathological data - a more complete picture of their animals.
The Histology and Tissue Processing Facility Core centralizes histology services providing routine processing, special techniques, technical assistance, training and consultation for Center investigators. The Core also makes necropsies at the request of members of the Center, particularly for the dissection of organs that may require special skills or knowledge of anatomy.
The Specific Aims of Facility Core 4 are:
- To utilize flow cytometry for multiparameter analysis of single cell suspensions and for isolation of purified subsets by cell sorting.
- To utilize confocal microscopy for precise localization of proteins and subcellular structures.
- To provide training and consultation on planning, executing and evaluating experiments involving flow cytometry and confocal microscopy.
Significance
The data obtained using the Cell and Tissue Analysis Facility Core benefits numerous Center investigators and supports research projects in all of the Research Focus Areas. Many of the Center members direct research projects that focus on the mechanisms by which environmental agents affect cellular survival, growth, differentiation and/or neoplastic transformation. Thus, it is imperative to evaluate the consequences imposed by environmental agents on the expression and stability of key proteins that influence these processes. Flow cytometry permits quantitative analysis of the changes in protein expression in individual cells and alterations in the distribution of distinct cellular subsets that ensue as a result of toxic damage or neoplastic transformation. Confocal microscopy allows these changes to be observed both at the subcellular level and at the level of tissue architecture. Furthermore, the ability to purify minor aberrant subsets by cell sorting based on qualitative or quantitative changes in protein expression level is a powerful tool for obtaining relevant target cells for subsequent molecular analysis.
Many of the Center investigators are also interested in the effects of various environmental agents and conditions on cell cycle progression in affected tissues. Flow cytometric analysis is important both for quantifying changes in the proportion of cells at various stages of the cell cycle and for sorting cells based on cell cycle stage. Similarly, flow cytometric analysis of apoptosis is an important tool that is used by several Center members who are interested in discerning the extent and kinetics of cellular survival in cells that have encountered environmental insults. Furthermore, flow cytometry is an ideal approach to identify a unique population of cells that differentially pumps out Hoechst 33342 dye using the p-glycoprotein pump thought to play a key role in multi-drug resistance. This property is considered to be characteristic of stem cell populations.
Further application details will be presented below. However, in general, the Cell and Tissue Analysis Facility Core provides Center members with the ability to assess the impact of environmental factors on cellular growth, differentiation and survival. Flow cytometry and confocal microscopy provide complementary methods of analysis. Flow cytometry of single cell suspensions provides rapid quantitative analysis of cellular subsets or constituents within a specified subset. On the other hand, confocal microscopy offers the ability to visualize tissue architecture and assess changes in the localization of cells or subcellular constituents. These approaches have played essential roles in the development and progress of numerous research projects within the Center.
Staff
Ellen Richie, Ph.D., Director
Kaoru Kiguchi, Ph.D., Co-Director
Kent Claypool, B.S., Flow Cytometry Facility Manager
