Flat-embedding

Aides L, Boone T. A combined flat-embedding, HRP histochemical method for correlative light and electron microscopic study of single neurons. J Neurosci Res 1984 11 27-34. 

Reymond O, Pickett-Heaps J. A routine flat embedding method for electron microscopy of microorganisms allowing selection and precisely oriented sectioning of single cells by light microscopy. J Microsc 1983 130 79-84. 

Sawada H, Esaki M. A simple flat embedding method for the correlative light and electron microscopic immunocytochemistry. J Electron Microsc 1998 47 535-537. 

The bound peroxidase is visualized by reaction with a filtered solution of 0.05% DAB and 0.0005% hydrogen peroxide in PBS. After rinsing with 0.15 M sodium phosphate buffer (pH 7.4), the sections are postfixed with 0.25% osmium tetroxide in the same buffer for 1 hr and counterstained with 1% uranyl acetate in deionized water. The sections are dehydrated and flat-embedded in Epon according to standard procedures (Hayat, 2000a). Controls are processed as above, except that incubation in the absence of the primary antibody is carried out with PBS containing 2% normal serum and 0.015% Triton X-100. 

For the electron microscopical inspection of microinjected cells, cover slips were fixed for 30 min in cacodylate puffer (50 mM cacodylate, pH 7.2 50 mM KCl, 2.5 mM MgCl2) containing 2.5% glutaraldehyde. The cells were then post-fixed with 2% osmium tetroxide (in 50 mM cacodylate, pH 7.2 15 min) and washed in H2O (30 min). After overnight staining with 0.5% uranyl acetate (in H2O), the cover slips were dehydrated in ethanol and flat embedded in Epon. 

A variety of molds are available to encapsulate samples for embedding. BEEM type capsules are useful for small pieces of materials that can be placed in the tip which then provides pretrimmed specimen blocks. Gelatin capsules are rounded in shape and thus must be trimmed, but they have the advantage that they can be removed by soaking in water. Other shapes are good for specific sample forms. Flat embedding molds are excellent for films and membranes. 

BEEM flat embedding mold (package of 12 Ted Pella 111-22). The type of mold used may vary depending on the microtome model. This size and style works well for a Reichert-Jung microtome. 

HRP injection fixation - vibratome sectioning HRP histochemistry (black) HRP ICC (brown) flat embedding LM selection of area of interest re-embeddings ultrathin sectioning s TEM... 

HRP injection s fixation s vibratome sectioning s HRP histochemistry s flat embedding s LM s selection... 

On the following day, sections are flat embedded between a glass slide (to provide a flat surface) and two sheets of ACLAR plastic. The first sheet is the spacer sheet, while the other is the cover sheet (Fig. 1). On the spacer sheet, make a hole with a razor blade larger than the tissue section. Glue the spacer sheet on top of the slide with super glue (Fig. 1). Then, a few drops of... 

Fig.1 Schematic view of flat-infiltration and flat-embedding procedure for array tomography. The tissue is fiat-infiitrated on top of an inverted glass petri dish containing a few drops of LR White, then covered by a glass coverslip (upper panel), and infiltrated overnight at 4 °C. On the next day, the tissue is flat embedded between a glass slide and two sheets of ACLAR plasBc (lower panel). One of the plastic sheets is a spacer while the other Is a cover. The tissue section is deposited in the hole of the perforated spacer sheet that was previously glued to one side of the glass slide (/owerpane/). Then, a few drops of LR White are added to the tissue section, and then covered by the cover sheet (lower panel). The tissue is incubated at 55 X for 24 h for polymerization...

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