This article describes a method for preparing semithin and ultrathin sections of osmium tetroxide-fixed mouse brain tissue for microscopic examination. The process involves aligning section images with an atlas, excising regions of interest, and using staining techniques to enhance visualization.
Take an osmium tetroxide-fixed mouse brain section embedded in a resin block.
Obtain a section image and align it with a pre-prepared atlas image to identify the region of interest.
Mark the borders of this region on the block.
Heat the resin to soften it and excise the marked region.
Glue the specimen to a glass holding bar and trim it.
Using an ultramicrotome, prepare semithin and ultrathin sections.
Transfer the semithin and ultrathin sections onto glass carriers and nickel grids, respectively.
Stain the semithin sections with a dye that binds to nucleic acids, ribosome-rich cytoplasm, and the extracellular matrix.
Wash the sections and examine them under a light microscope to confirm the presence of the target area.
Observe the ultrathin sections under a transmission electron microscope.
Osmium tetroxide enhances the electron density of cellular and organelle membranes, providing high contrast against the less electron-dense cytoplasm, enabling visualization of cellular ultrastructure.
To map an area of interest, select an image containing the area of interest from the previously prepared image atlas. Sketch the borders of the area of interest onto the sectioned image. Optically superimpose these borders onto the embedded specimen under the microscope, and use a one-inch 26 gauge needle to scratch these region borders onto the resin specimen. Then, heat the specimens to 95 degrees Celsius in order to soften the resin.
Next, take out the warm resin specimens from the oven, and use a razor blade to excise the areas of interest. Glue the specimens onto acrylic glass holding bars of the appropriate caliber, and trim the mounted specimens for semi and ultra thin sectioning. Use an ultramicrotome to acquire semi thin 0.7 micrometer and ultra thin 70 nanometer sections, collecting the semi thin sections on glass carriers and the ultra thin sections on nickel grids.
Stain the semi thin sections with 1% toluidine blue in PBS for four minutes, followed by several washes in deionized water before examining the sections by light microscopy. The ultra thin sections can then be directly assessed by transmission electron microscopy at 180 kilovolts, without further modification.
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