The Anatomy and Histology Facility functions as an extension of the investigators' own labs. Researchers come to use the facility's microtomes; collaborate with facility staff doing animal anesthesia and perfusion; or request tissue sectioning and labeling with stains, antibodies, and DNA/RNA hybridization probes. For each preparation the staff members accommodate the specific needs of the labs they support.
The facility has 1,000 square feet of workspace and is equipped for tissue fixation, sectioning, immuno-labeling, and staining of a variety of mouse brains and other cells and tissues. Facility staff also prepare cultured brain slices and dissociated hippocampal neurons for physiology experiments. There are two chemical fume hoods for fixatives, a cell culture hood, a clean box, and four CO2 incubators for preparing primary cultures. Facility staff have extensive experience with antibody-labeling methods. Some investigators prefer to use the facility's equipment on their own, while others may request staff assistance in performing various histological procedures and methods.
- Cryostats and vibratomes. The facility has two Leica 3050S cryostats, five Leica vibratomes, and a CryoJane tape transfer system. All items are available to investigators for their own use.
- Tissue culture hoods. Culture hoods can be booked by researchers using an online scheduler.
- Microscopes. There is a Zeiss inverted fluorescence microscope for examining primary cultures and an Olympus MVX10, a fluorescence dissecting microscope, for inspecting GFP and antibody labeling in sections.
Services and Training:
- Anesthesia and perfusion-fixation of mice and rats
- Embedding in OTC and agarose
- 5 µm-thick to 50 µm-thick sections prepared on Leica cryostat or vibratome
- Antibody labeling and histochemical staining (Nissl, H&E, special stains)
- Primary cultures of hippocampal neurons
- Preparation of brain slice cultures for physiological recording
- In-situ hybridization
Through services, training, and collaborations, the Cell and Tissue Culture Facility acts as a stimulating environment for scientific interaction among users and staff, and provides cultures for cell-based assays and tool development.
Cell Culture Services:
We provide services in cell culture and cell transfection in a 1,000 square foot facility equipped with nine culture hoods and 15 incubators. The shared resource provides quality control of equipment, reagents, and cell stocks and performs cell transfection using a variety of reagents and protocols. The staff members maintain a variety of cell lines, performs culture expansion and storage. Half of the facility space and equipment is designated for researchers who want to culture and transfect cells on their own. The shared resource maintains two separate BSL2 room for culturing viruses, such as adeno-associated virus (AAV) and lentivirus.
Flow cytometry capabilities are provided by the Cell Culture Shared Resource which operates a Becton Dickinson FACSAria flow cytometer. The FACSAria is a high speed sorter with a fixed-alignment cuvette flow cell capable of sorting 25,000 events per second for lymphocytes and approximately 5-8,000 events for larger cells. Our instrument is a special order FACSAria that contains 5 lasers, 20 fluorescent detectors, 4 nozzle size options, and optical filters to detect approximately 17 different fluorophores. It can sort four populations simultaneously and we have the capability of sorting into multi-well plates or onto slides.
Recombinant Protein Production:
The Cell Culture Facility provides recombinant protein production using baculovirus, yeast, bacterial and mammalian expression systems. Other related capabilities include vector construction, western blot confirmation of expression and protein purification.
• Fast-turnaround DNA sequencing using a 16 channel ABI 3130xl Genetic Analyzer
• DNA isolation services for all scales of plasmid DNA, as well as BAC, YAC, or cosmid
• Rapid small-scale AAV production
• Illumina HiSeq 2500 for next-generation sequencing applications
• Quantitative PCR for genotyping and RNA analysis
• Vector design and construction and development -- either finding the right vector from existing sources or engineering unique vectors specifically for your purposes. The Facility also has the capacity to produce clone collections using these vectors.
• Molecular biology enzyme freezer
Concerning sequencing protocols, the Facility can do low-throughput sequencing (and select rush jobs) in-house or can arrange to have high-throughput large volume analyses done through outside vendors.
The Molecular Biology Facility maintains and operates an ABI 3130xl DNA analyzer. This is a 16-capillary machine, capable of processing a maximum of ~384 sequencing reactions over 24 hours of continuous operation. The Facility also offers specialized sequencing services, such as transposon-mediated sequencing and direct sequencing from colonies using rolling circle amplification (RCA). Researchers in need of higher-volume sequencing should contact the Facility manager about outsourcing options.
DNA Preparation & Analysis
The Facility provides a DNA isolation service for purifying plasmid, BAC, YAC, phage and cosmid DNA. Endotoxin-free plasmid DNA preparations are available. Other methods are available upon request. The Facility performs rolling circle amplification (RCA) for rapid enzymatic amplification of DNA that eliminates the need for overnight culturing and minipreps for sequencing applications. The Facility can also provide post-purification services, such as restriction enzyme analysis, sequencing, and purification of inserts for microinjection.
AAV and Lentivirus Production. The shared resource produces quick turnaround small-scale preparations of adeno-associated virus (AAV) at a minimum titer of 10^12 (GC/ml). The available serotypes include 1,2,5,7, and 9. We also offer production services for lenti- and rabies virus.
Next Generation Sequencing
The facility provides next generation sequencing (NGS) using the Illumina HiSeq 2500 platform. Technical assistance is available for RNA isolation, and RNA-Seq library construction. Vector Design and Development
The Facility provides a complete cloning and vector construction service for the production of expression, reporter, lentivirus or AAV, or transgene constructs and functional testing. We have experience with utilizing Gateway and recombineering techniques for higher throughput cloning applications.
The facility maintains a MX3005P real-time PCR instrument.
Verification of transgenes through PCR based genotyping and sequencing is provided.