- About CURE
Imaging and Stem Cell Biology Core (ISCB)
The overall goal of the Core is to enhance the effectiveness of the CURE: DDRCC program by providing cost effective services through centralized resources and facilities, as well as training and assistance for the application of morphological, imaging and stem cell biology technologies and promoting collaborations among CURE:DDRCC investigators with independently-funded research projects requiring these approaches. This goal will be achieved by the following specific aims:
1) To provide educational resource, training and expertise for the visualization of chemical messengers and proteins at the cellular level and characterization of phenotypical aspects of genetically engineered mice using histological and immunohistochemical approaches, and access to a bank of highly characterized polyclonal and monoclonal antibodies.
2) To provide training, assistance and access to state-of-the-art equipment for an array of contemporary imaging approaches to facilitate investigations in trafficking and signaling of receptors and their ligands, and subcellular localization of signaling molecules.
3) To provide training, facilities and expertise for the preparation and isolation of human and murine intestinal stem cell, and to generate 3D in vitro culture systems, which can be used to help model human epithelial and smooth muscle disorders.
Overall these aims will facilitate advancement in basic and translation research in digestive diseases.
The Core personnel will train individual investigators or their staff, then they will be available for consultation, once the investigators are sufficiently comfortable and familiar with the methodology and experimental design for their own investigations.
A. Tissue Preparation for immunohistochemistry or histology:
- Tissue and Cell Fixation according to the cell type and the antibodies or staining;
- Tissue Embedding and Sectioning, including frozen sections using cryostat or microtome, or paraffin method for structural analysis and localization studies;
- Whole mounts, and organotypic and cell cultures. Whole mounts provide for a better view of the distribution and accurate quantification of cell bodies. Organotypic cultures allow studying receptor distribution and trafficking in tissue with preserved neuronal networks. Primary cell cultures allow studies of receptor trafficking and signaling at the cellular level and transfection with lentiviral vectors prepared by the Integrated Molecular Technologies Core.
B. Immunohistochemistry for the following purposes:
- Localization of different molecules (e.g. peptides and receptors) using single and multiple fluorescent labeling or immunoperoxidase staining;
- Investigation of activated receptor trafficking by visualizing ligand-induced receptor internalization,
- intracellular sorting and recycling in transfected and native cells using immunohistochemistry and confocal microscopy.
C. Monoclonal and polyclonal antibodies central bank. Antibodies generated at CURE during previous
funding periods will be made available to CURE: DDRCC members and to other investigators outside the Center. These antibodies most often are used for immunohistochemistry and Western blot, but some have been validated for immunoneutralization studies.
D. Light Microscopy, High Resolution Fluorescence Imaging with DigitalPhotography, and Image Analysis for transmitted and fluorescent microscopy with microscopes equipped with high resolution cameras for capturing high quality images and for quantitative determinations of phenotypic changes in gene expression, morphometric analysis and receptor quantification in different cellular compartments.
E.UPDATED SERVICE: Confocal Microscopy for the 1) intracellular signaling pathways, 2) cellular and subcellular localization of molecules, and 3) definition of spatial relationship between
receptors and their ligands. The recently acquired Zeiss LSM 710 with Axioplan 2 microscope for live and fixed cell imaging is a state-of-the-art confocal microscope with highly efficient, affinity corrected optics, multi-channel detectors, and user-friendly software with wide range of functions including two separate 3-D reconstruction options. Images captured at the confocal microscope, can be analyzed at other substations using software made available at the other locations.
F. UPDATED SERVICE: Fluorescence Analysis of Living Cells. This includes different videofluorescence approaches that allow studying events at the cellular level, including:
F1. UPDATED SERVICE: Ca2+ imaging with imaging GFPs and other intracellular fluorescent probes for the analysis of signal transduction pathways in single cells using 1) the Zeiss AttoFluor Ratio vision with Axiovert 135 microscopes, quartz optics and a temperature control perfusion chamber with an Eppendorf microinjection system to introduce non-permeant dyes into cells for the visualization of intracellular events, 2) a Zeiss Pascal confocal mounted on an upright Zeiss Axoplan 2 microscope equipped with both short wave (405 nm) and visible wavelength lasers for excitation for whole cell patch clamp recording simultaneously with confocal live cell Ca2+ imaging and 3) a Zeiss high speed, high resolution imaging system consisting of a high sensitivity high resolution camera which can be used for the detection of different intracellular indicators of signaling with different fluorophores and at different locations in the cell
F2. Photoactivatable proteins, such as photoactivatable GFP (PAGFP), Dronpa and Kaede, for monitoring the intracellular movement of molecules (kinases, receptors and proteins) shuttled
between the nucleus and cytoplasm in living cells with high temporal and spatial resolution. This
methodology, with our Zeiss Pascal confocal imaging system, allows analysis of real time distribution of a region of interest.
J. NEW SERVICE: Isolation and In vitro culture methods of Human and Murine Small and Large Bowel Epithelium, smooth muscle cells and myofibroblasts are species specific methods routinely
performed in Dr. Martin’s lab. Isolated cells can be used to perform 3D culture or fluorescence-activated cell sorting (FACs). These protocols can be used to isolate, process and grow intestinal epithelium, enteroids and other mesenchymal cells. Various co-culture methods, including the
utilization of various matrices for the 3D culture system such as Matrigel and a pure collagen-based method that work in human and murine samples have been developed and refined (Figs. 5A &B).
K. NEW SERVICE: In vitro retroviral transduction methods of Human and Murine Small and Large
Bowel Epithelium, and mesenchymal cells that can be used to alter expression of various genes and
pathways by transducing lentiviruses that modulate expression and genes and pathways
Catia Sternini, MD
Imaging and Stem Cell Biology (ISCB) Core Director