With in situ molecular mapping techniques we can explore the particular functions developed by cell structures and how macromolecular complexes build organelles and cells. In the lab we mainly work with two groups of methods: immunogold labeling (on plastic resin sections or Tokuyasu cryosections) and labeling with clonable tags for electron microscopy and CLEM (correlative light and electron microscopy).
- Immunogold labeling on Tokuyasu cryosections. The Tokuyasu technique is mainly used for immunohistochemistry. This technique avoids dehydration and infiltration with a plastic resin. Samples are chemically fixed with aldehydes, cryo-protected with sucrose and frozen to harden them before thin sectioning. Thin sections are then thawed at room temperature and transferred to formvar/carbon-coated grids. The sections can be labelled with any affinity probe and a probe linked to colloidal gold before embedding in a mixture of uranyl acetate and methylcellulose that provides contrast and protection during drying. This technique yields an excellent visualization of membranes together with an optimal preservation and labelling of macromolecules (I. Hurbain & M. Sachse, Biology of the Cell, 2011).
- Clonable tags for EM and CLEM. METTEM (Metal Tagging Transmission Electron Microscopy) is a method developed in our laboratory to detect proteins in cells with high sensitivity. The procedure uses the small metal-binding protein metallothionein (MT) as a clonable tag. When fused with a protein of interest and treated in vivo with gold salts a single MT tag will build an electron-dense gold nanocluster ~1 nm in diameter, easily detectable by transmissible electron microscopy. When proteins are double tagged with green fluorescent protein (GFP) and MT, direct correlative light and electron microscopy allows visualization of the same macromolecular complexes with different spatial resolutions (I. Fernández de Castro, L. Sanz & C. Risco, Methods in Cell Biology 2014).
Methods for in situ molecular mapping
With in situ molecular mapping techniques we can explore the particular functions developed by cell structures and how macromolecular complexes build organelles and cells. In the lab we mainly work with two groups of methods: immunogold labeling (on plastic resin sections or Tokuyasu cryosections) and labeling with clonable tags for electron microscopy and CLEM (correlative light and electron microscopy).
- Immunogold labeling on Tokuyasu cryosections. The Tokuyasu technique is mainly used for immunohistochemistry. This technique avoids dehydration and infiltration with a plastic resin. Samples are chemically fixed with aldehydes, cryo-protected with sucrose and frozen to harden them before thin sectioning. Thin sections are then thawed at room temperature and transferred to formvar/carbon-coated grids. The sections can be labelled with any affinity probe and a probe linked to colloidal gold before embedding in a mixture of uranyl acetate and methylcellulose that provides contrast and protection during drying. This technique yields an excellent visualization of membranes together with an optimal preservation and labelling of macromolecules (I. Hurbain & M. Sachse, Biology of the Cell, 2011).
- Clonable tags for EM and CLEM. METTEM (Metal Tagging Transmission Electron Microscopy) is a method developed in our laboratory to detect proteins in cells with high sensitivity. The procedure uses the small metal-binding protein metallothionein (MT) as a clonable tag. When fused with a protein of interest and treated in vivo with gold salts a single MT tag will build an electron-dense gold nanocluster ~1 nm in diameter, easily detectable by transmissible electron microscopy. When proteins are double tagged with green fluorescent protein (GFP) and MT, direct correlative light and electron microscopy allows visualization of the same macromolecular complexes with different spatial resolutions (I. Fernández de Castro, L. Sanz & C. Risco, Methods in Cell Biology 2014).