The facility provides advanced expertise in electron microscopy, from sample preparation to image analysis, for a wide variety of biological samples.
Researchers are frequently looking for tools to localise proteins of interest within biological samples. Specific antibodies, revealed by fluorescent probes, are frequently used to solve these questions at the light microscopy level.. To resolve the intracellular structure together with the immunolabeling pattern at higher resolution with an electron microscope, antibody staining can be revealed with colloidal gold particles. . For such an Immuno-EM experiment, samples are chemically fixed and embedded in a support layer. Ultrathin sections of 70 nm are cut and immunolabeled with the antibody of choice followed by a binding step with an electron dense Gold particle (visible in the EM).
An efficient immunolabeling procedure is the Tokuyasu cryo-section technique. With this technique, a biological sample is fixed with mild chemicals such as glutaraldehyde and paraformaldehyde directly or by High Pressure Freezing and Automatic Freeze Substitution). After fixation the sample is embedded in a gelatin support, infused with sucrose and frozen in LN2. Thin sections are cut in a ultracryotome at -120˚C using a diamond knife. Sections are retrieved, thawed, placed on EM-grids and immunolabeled followed by EM analysis. Unfortunately, high antigenicity only goes together with mild fixation and therefore good morphology is sacrificed.
Other substrates like acrylic resins (Lowicryl) have been used as well for immunolabeling. The advantage is the better morphology over the Tokuyasu method, but the labelling efficiency is much lower. Crosslinking of the resins will hide antigens and prevent binding to antibodies.
Advanced IEM
Beside a single antibody labelling there are ways to perform double-, or even triple immunolabeling procedures to identify 2 or 3 proteins of interest in one section.
Immuno Correlative Light Electron Microscopy (iCLEM) is a strong tool to find rare events at high resolution while keeping ultrastructural context intact.
Both advanced methods are challenging and well-defined control experiments should be taken into account. Validation of antibodies is always a must when performing immunolabeling experiments.
Ongoing Projects:
The flag tagged NOX4 enzyme is expressed in the endosomal system in kidney proximal tubule cells. A specific anti NOX4 and anti Flag are used to identify the organelles of interest.
Neuronal cells in Optic Chiasma of mice are identified with viruses expressing GFP fused with an axonal tag or fused with a synaptic protein. Immunolabeling done with anti-GFP.
VAP-A is characterised in primary Mesenchymal Stem Cells by an antibody against VAP-A.
References:
Slot J., Geuze H. Cryosectioning and immunolabeling. Nat Protoc. 2007;2(10):2480-91.