Tissues are preserved in formaldehyde to allow the best ultrastructural detail, and if needed, to balance detail with immunoreactivity.
Tissue processing in epoxy resin, acrylic resin or polyester wax
- Methods are customized for the particular project, using epoxy resin for ultrastructural detail and pre-embedded immunolabeling, and acrylic resin for on-grid immunogold labeling. Light microscopic immunohistochemistry is often used to guide the ultrastructural immunolabeling approach, and polyester wax-embedded tissue is frequently more sensitive to antibody reactions than paraffin-embedded tissue.
- Semi-thin sections (0.5 µm) of acrylics and epoxies are placed on glass slides and stained with toluidine blue for light microscopy review.
- Ultrathin sections (50-70 nm) of epoxies are placed on EM grids for ultrastructural transmission electron microscopy, while ultrathin sections of acrylics on EM grids are used for immunogold labeling.
- Vibratome sections may be 50 mm or thicker and used for immunoperoxidase or immunogold labeling of free-floating sections before they are embedded.
- Polyester wax or paraffin are usually cut 2-3 µm thick for immunohistochemistry and other light microscope procedures.
Immunoperoxidase and immunogold labeling
- Tissues are preserved and processed to balance immunoreactivity with ultrastructural preservation. Methods are customized for a particular project. Options include pre- and post-embedding methods, using either acrylic embedded samples for on-grid immunogold labeling, free-floating vibratome sections of preserved tissues for pre-embedding immunoperoxidase or immunogold labeling, or on-grid labeling of ultrathin cryosections. Light microscopic immunohistochemistry is often used to guide the ultrastructural immunolabeling approach.
- On-grid immunogold label of acrylic-embedded ultrathin sections. Pre-embedding immunoperoxidase and immunogold labeling of free-floating vibratome sections or cultured cells, and on-grid labeling of ultrathin cryosections or direct preparations of samples are other strategies we use.
- Light microscopic immunoperoxidase correlated with ultrastructural immunogold: Immunohistochemical staining of kidney for ferroportin (left panel) and immunoelectron micrographs of the proximal tubule, showing ferroportin immunogold labeling in the proximal tubule brush border (right panel, arrows). From Bolisetty, et al., J. Clin. Invest. 2013 Oct 1;123(10):4423-34
- Uranyl acetate and lead citrate are used on samples for electron microscopy to provide contrast in the tissue.
- The Hitachi 7600 transmission microscope and the Nikon bright field microscope are both equipped with digital imaging systems.
- Quantifies cell area, organelle area or volume density, and plasma membrane boundary length or surface density using stereologic methods to characterize specific cells or determine the effect of experimental maneuvers on cell characteristics.
- Combines morphometric analyses of ultrastructure with quantified immunogold label to determine cell-specific and subcellular abundance and relative distribution of immunolabel
Application of stereological principals to quantify changes in cell structures alone, or in combination with immunogold to quantify changes in specific protein cellular expression and subcellular distribution. Electron micrographs (above) show metabolic acidosis increased apical plasma membrane, decreased subapical cytoplasmic vesicles, and redistribution of the ammonia transporter, Rhcg, from the cytoplasm to the apical plasma membrane of type A intercalated cells in rat kidney. The changes in structure and Rhcg distribution were quantified and illustrated graphically. Seshadri R M et al. Am J Physiol 2006;290:F1443-F1452
Submissions are handled first come, first served with every attempt to have work finished as soon as possible. Please let us know about deadlines so we can try to meet them. If you need a quicker turnaround time, please ask staff to expedite. Additional fees may apply.
Here are some approximate times but it also depends on current workload as well as the complexity of your submission
- Tissues for polyester wax processing and embedding only: 1-3 days.
- Tissues for electron microscopy, processing and embedding in acrylic or epoxy: 3-5 days.
- Paraffin or polyester blocks to unstained slides: depending on number of blocks, number of slides/block, and any special requests (multiple sections/slide, multiple blocks/slide) 1 or more days
- Epoxy and acrylic semi-thin sections, 3-4 sections/slide, stained with Toluidine Blue, 1-3 days.
- After the investigator examines the semi-thin sections and determines where to cut ultrathin sections, it will be an additional 2-3 days for sectioning the ultrathins, placing them on grids, and counterstaining.
- Immunohistochemistry or immunogold usually takes 2-3 days or longer, depending on whether the antibody is under development or has been routinely used.
When samples are ready, you will be notified by email or phone. You may then make an appointment to use the microscope, or to pick up your slides.
Acknowledgement of specific instrument use and/or core service is a metric tracked by both the institution and NIH as a measure of core productivity. Please consider acknowledging the core and/or staff in your publications as:
“…the staff of the University of Florida College of Medicine Electron Microscopy Core Facility”, and, if applicable, the name of the staff member who provided you with advice and technical assistance.