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A simple shortcut for observing unroofed cells by either TEM or SEM
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DOI: 10.1002/9783527808465.EMC2016.5846. International audience. The “unroofing” technique has been successfully used to observe the cytoplasmic side of the plasma membrane (PM) using either light or electron microscopy. Combined to transmission electron microscopy (TEM), it is an invaluable method to reveal the composition of the PM and to directly observe macromolecular complexes including the cytoskeleton and endocytic membrane invaginations. This method has been optimized over decades to preserve membranes close to their native states by the combination of quick freezing of exposed membranes, followed by deep etching and rotary replication (the so-called “QF-DE-RR” technique). However, a serious setback in implementing unroofing combined with QF-DE-RR stems from the necessity to use complicated apparatus, such as quick freezing and freeze-fracturing devices, along with strong expertise to handle them. Moreover, the technical complexity renders these techniques time consuming and reduces the number of samples that can be processed simultaneously. Here, we present a simple and straightforward protocol for observation of the cytoplasmic side of plasma membrane which only requires chemical treatment of samples prior to replication This method has been optimized towards sample preparation at room temperature, chemical fixation, dehydration, solvent drying and sequential metal coating. Moreover, this technique is easily amenable to higher throughput. We compared either TEM or high resolution SEM analysis of unroofed membranes from adherent cells and show the advantages and disadvantages of each technique towards visualization of the cytoskeleton and different endocytic structures such as clathrin coated pits and caveolae.
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