Cellular and In Vivo Models Core

Overview

The Cellular and In Vivo Models Core serves the CSIBD community by providing Center investigators with the technical support, expertise, and access to high-end instrumentation necessary for morphological, immuno-cytochemical, and cell biological studies related to digestive disease and IBD research. Additionally, the core offers expert advice for the generation, implementation, and analysis of in vitro and in vivo model systems during investigations of genes and pathways that yield insight into cellular networks contributing to IBD and other digestive diseases.

Objectives

1. Provide access to sophisticated cellular imaging approaches at a time when few or no isolated laboratories can develop the highest level of technical competence in the specialized areas served by the Core
2. Enable transformative investigation into the impact of human genetic variants associated with IBD by providing advice on in vivo model systems, access to a genotyped organoid bank, resources for manipulating organoids and primary epithelial cells, and histopathological analysis
3. Provide an environment appropriate for the training of key personnel from participating laboratories
4. Evolve to meet the changing needs of the research community by introducing new equipment and techniques
5. Maintain the highest level of integrity in generating, analyzing, and reporting data by focusing on quality control, rigor and reproducibility parameters in research, and data management
6. Provide a central location for imaging activities in which different investigators can interact and share ideas for pushing IBD research forward

Services

• Routine tissue fixation and processing for light microscopy. The Core also performs special stains (e.g., trichrome, PAS, Sirius Red).
• Perfusion-fixation of organs. The type of fixative depends on the experiments to be carried out and sensitivity of antigens to fixation; it is determined empirically based on our experience.
• Conventional electron microscopy. Images are captured digitally using our JEOL 1011 electron microscope and AMT CCD camera. They are stored on a central file server.
• Low temperature embedding in Lowicryl HM20. Embedding tissues in hydrophilic resin preserves antigenicity better than conventional embedding and allows the post-embedding detection of antigens on thin sections of tissues, using immunogold labeling techniques.
• Immunocytochemistry. The Core performs and instructs users on a variety of immunocytochemical procedures, including the following:
• Light microscope immunocytochemistry on cells and tissue sections with 3D reconstruction
• Light microscopy of intact cells and tissues
• Electron microscopy on intact cells and tissues (pre-embedding labeling)
• Immunogold electron microscopy on thin sections
• Ultracryomicrotomy for immunogold labeling of thin, frozen sections
• Nikon A1R laser scanning confocal microscopy. This is an invaluable tool for pursuing a variety of studies on cells and tissues with sophisticated techniques such as FRET and FRAP, real time and spectral imaging
• Zeiss LSM800 Airyscan confocal imaging. Our new confocal system allows image capture at close to double the resolution and much greater sensitivity compared to conventional confocal systems.
• Opal multiplexed imaging (Perkin Elmer). This is a new technique that allows up to seven antibodies (even if they are all raised in the same species) to be used for labeling. Each step results in covalent crosslinking of a specific fluorophore with the target antigen, followed by stripping of the antibodies, and re-labeling with the next antibody – up to seven times. It requires a confocal, such as our Nikon A1R system, that is capable of precise spectral imaging to distinguish the seven probes with no overlap.
• Spinning disk confocal microscopy. The Perkin Elmer spinning disk confocal microscope is designed for close to real-time acquisition of images from live cells or other small specimens.
• Total internal reflection fluorescence (TIRF) microscopy. TIRF is a powerful technique for visualizing fluorescently labeled molecules that are in or near the cell membrane.
• Calcium/pH ratio imaging. We have adapted our TIRF microscope to perform Ca++ and pH ratio imaging.
• Nikon STORM (super-resolution) microscopy. This system is a form of single molecule light microscopy that enables the visualization of interactions, such as between proteins or between proteins and nucleic acids, at the nanoscopic level. STORM capabilities include the use of three-color and three-dimensional imaging.
• Multiphoton confocal microscopy via the MGH Center for Systems Biology (CSB). The Program in Membrane Biology is within the MGH Center for Systems Biology. Interested users from the CSIBD may gain access to these Olympus systems by contacting Dr. Dennis Brown.
• Imaging flow cytometry. Amnis technology fully distinguishes and ­­visualizes appropriate information from undesired background debris by a combination of image analysis and brightfield examination.
• Central antibody bank. We have a bank of close to 2,000 antibody reagents, many of which were developed in-house.
• Personnel training. We provided support for CSIBD investigators by (1) consultation activities, (2) short-term technical training, and (3) intensive training of selected personnel in specific activities that will be used extensively by participating laboratories.