methylcellulose and uranyl-acetate

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Topics: Lipids; Liposomes; Metals, Heavy; Methylcellulose; Microscopy, Electron, Transmission; Nanoparticles; Negative Staining; Organometallic Compounds; Phosphotungstic Acid; Silicates; Specimen Handling; Staining and Labeling; Tungsten Compounds

One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.

Topics: Animals; Citrates; Cryoultramicrotomy; HL-60 Cells; Humans; Lysosomal-Associated Membrane Protein 2; Methylcellulose; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Muscles; Muscular Dystrophy, Facioscapulohumeral; Organometallic Compounds; Osmium Tetroxide; Staining and Labeling; Tissue Embedding

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

Topics: Animals; Autophagy; Carbonic Anhydrases; Cathepsin D; Cells, Cultured; Cryoultramicrotomy; Endocytosis; Endosomes; Immunohistochemistry; Liver; Male; Methylcellulose; Organometallic Compounds; Rats; Rats, Wistar; Superoxide Dismutase; Vacuoles

Ultra-thin frozen sections are ideal substrates for immunolabelling in high resolution electron microscopy. However, visualization of subcellular structures is inferior to that obtained with corresponding plastic sections. Although negative staining is generally effective and even superior to positive staining, the accumulated stain is often too heavy, obscuring morphology and markers used for immunocytochemical localization of antigens. This paper describes the development of a modified negative contrast staining technique in which a high concentration of uranyl acetate is mixed with methyl cellulose at a low pH. Application of this stain to cryosections of cells and tissue resulted in improved visualization of morphological structures characterized by negative images of membranes and cell organelles. Use of this stain is advantageous for morphological and immunocytochemical studies involving ultra-thin frozen sections.

Topics: Adrenodoxin; Animals; Cell Line; Chickens; Female; Frozen Sections; Granulosa Cells; Hydrogen-Ion Concentration; Kidney; Methylcellulose; Microscopy, Immunoelectron; Microtomy; Muscles; Organometallic Compounds; Rats; Rats, Wistar; Spleen; Staining and Labeling

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.

Topics: Acrylic Resins; Adsorption; Animals; Frozen Sections; Histocytochemistry; Histological Techniques; Humans; Methylcellulose; Microscopy, Electron; Organometallic Compounds; Rats; Staining and Labeling