Finally, FISH probes are hybridized to the anchored RNA.
The samples then go through gelation, mechanical homogenization, and expansion. Samples are fixed and treated with LabelX (see panel E, right, for detail), which enables RNA to be anchored to the polymer. (D) Expansion fluorescence in situ hybridization (ExFISH). (C) Samples are fixed and treated with AcX before going through gelation, a comparatively gentle mechanical homogenization process (e.g., high temperature denaturing in detergent solution), and expansion, followed by antibody staining. (B) Samples expressing fluorescent proteins are fixed and treated with AcX before going through gelation, mechanical homogenization, and expansion in water. The samples then undergo gelation, proteinase K (ProK) treatment for mechanical homogenization (also called digestion), and expansion in water. A, Samples are fixed and stained with antibodies using conventional immunostaining protocols, then treated at room temperature (RT) with Acryloyl-X SE (AcX, see panel E, left, for detail), which enables proteins to be anchored to the hydrogel. (A–C) Protein retention ExM (proExM) workflows. Utilizing a lens with 300 nm diffraction limited resolution, the resulting ~4.5x linear expansion of the embedded samples (~90x volumetric expansion) allows imaging of the sample with ~300 nm / 4.5 ~ 60–70 nm effective resolution.Įxpansion microscopy (ExM) workflows discussed in this unit. Fluorescent proteins and antibodies are sufficiently resistant to proteolysis that they survive the ProK application. 1A, or pre-expansion fluorescent protein expression, Fig.
One can alternatively apply AcX to fixed specimens expressing fluorescent proteins and/or labeled already with fluorescent antibodies, incorporating these directly to the gel (pre-expansion staining, Fig. This version uses a mechanical homogenization method employing high temperature and detergent, without protease. One can anchor native proteins to the gel using AcX and apply fluorescent antibodies post-expansion (post-expansion staining, Fig. An enzymatic digestion using proteinase K (ProK) mechanically disrupts the embedded sample, allowing for isotropic expansion in water. 1E, left), and is then incorporated into the hydrogel polymer during the process of polymerization (also known as gelation for a hydrogel) (schematized in Fig. In proExM, proteins are covalently anchored to a hydrogel matrix with a commercially available small molecule (Acryloyl-X SE, or AcX for short) ( Tillberg et al., 2016) that binds to primary amine groups on proteins ( Fig. This unit describes the most common scenarios for proExM and ExFISH sample preparation, as well as general guidelines for sample handling, and image acquisition.
Recently, two new ExM versions have been developed, termed protein retention expansion microscopy (proExM) ( Tillberg et al., 2016), and expansion fluorescence in situ hybridization (ExFISH) ( Chen et al., 2016), where protein (proExM) and RNA (ExFISH) molecules are chemically anchored to the hydrogel and probed upon expansion. In expansion microscopy (ExM), preserved biological samples are embedded within a swellable hydrogel and physically expanded isotropically, leading to optically transparent samples which allow for nanoscale resolution and aberration-free microscopy imaging on conventional diffraction limited microscopes ( Chen et al., 2015). Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light sheet microscopes is provided. Here, we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence in situ hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ~4.5 fold linearly when immersed in water. Expansion microscopy (ExM) is a recently developed technique that enables nanoscale resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes, via isotropic physical expansion of the specimens before imaging.
0 kommentar(er)
