Within this section we will present some basic techniques that are applicable to most histological studies, which can be appropriately categorized as fixation, sectioning, and embedding. Since these techniques are not specific to neurohistology, they will only be briefly described.
Modern Techniques of Neuroscientific Research
Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Operation method
Primary endpoints of mammalian muscle shuttles-experiments using light microscopy with 1 um continuous sections
Materials and Instruments
Cat Partially exposed muscle shuttle Move I. Fixed Removed muscles were fixed in situ for 5 min in 0.1 mol/l sodium dimethylarsenate buffer containing 5% glutaraldehyde at pH 7.2 (glutaraldehyde is often diluted in 25% solution). (Glutaraldehyde is often diluted in 25% solution and should be stored below 4°C before use because it polymerizes easily). The muscle fibers were cut into 10 mm lengths to ensure that at least one myofibril was present, and fixed in the same fixative for another 4-14 d. (The total fixation time varied from laboratory to laboratory, and differences in fixation time did not generally affect the fixation effect significantly.) Wash with buffer for 30 min. Osmium tetroxide is fixed in 1% osmium tetroxide prepared in buffer for 4 h. (Osmium tetroxide penetrates slowly, but the thickness of the thin, short, cylindrical muscle is less than 1 mm, so 4 h is sufficient for adequate fixation. Osmium tetroxide is usually prepared as a 2% storage solution, bottled and sealed, and stored in a refrigerator. To prepare the fixative, an equal amount of 0.2 mol/L sodium dimethylarsenate buffer was added to the fixative to dilute it to the desired final concentration). The specimens were dehydrated with graded ethanol at 70%, 95% and 100% (twice) for 10 min each time at room temperature. The specimens were treated with a 1:1 mixture of ethanol and propylene oxide (1,2-epoxypropane) for 15 min [Propylene oxide is commonly used as an intermediate solvent and acts like a "hyaluronan" in paraffin embedding methods. The refractive index of most thylakoids is similar to that of dehydrated proteins and other intracellular components. The name comes from the fact that they were originally used to immobilize transparent tissues, and although it is now known that they have little or no such function, the name has stuck. For other methods of dehydration see Glauert (1975)]. Epoxy-propylene burnishing treatment for 15 min. Soak with a 1:1 mixture of propylene oxide burnt with Epon (ingredients other than accelerant) and place in an open container in a fume hood overnight (volatile propylene oxide helps the sclerosing agent to penetrate better into the tissue mass). Pour off the remaining osmotic medium in the bottle and transfer to fresh, pure Epori. The slices were flatly embedded in aluminum foil-lined molds and polymerized at 45°C for 12 h and then at 60°C for 24 h. The slices were then polymerized at 60°C for 24 h. Sections are made manually with the aid of a conventional glass knife using an ultrathin slicer with a slice thickness of 1/um, and every 10 slices are collected in groups of 1. (If necessary, the slices may be laid flat on the water surface either by approaching the slices with a chloroform-vaporized brush or by applying radiant heat through an electric heater. The glass knife should be replaced frequently, with care taken to reset the knife accurately when replacing it, and a mechanical switch should be used to control the movement of the knife to ensure that the same section is obtained for successive cuts. Since the distance between the knife and the surface of the tissue block is only a few micrometers, in order to still position the knife accurately after replacing it with a new one, the back of the knife can be illuminated, at which point the gap between the knife blade and the surface of the tissue block appears as a bright line). The coverslip (standard size 50 mmX20 mm) is cut into narrow strips 3 mm wide with a diamond knife, and the sections are collected directly from the sink by dipping the end of the narrow strip under the surface of the liquid (Fig. 1-1A) (these sections are either arranged in bunches or individually. A toothpick with an eyelash attached can easily be used to introduce the slice onto the surface of the narrow strip, which should be held with clockwork forceps. The narrow strip is held with clockwork forceps, and it is easy to adhere the section to the narrow slide by placing one side of the strip close to the tongue-like protruding end of the section and then drying the section (this method is easy and results in a good adhesion of the section to the narrow slide). Dry the back of the narrow strip with a soft fabric and allow the section to float in the water droplets at the front of the narrow strip. Use a hot plate at a temperature of about 70 "C to dry the slice and attach it to the surface of the narrow strip [it is best to permanently attach a glass slide to the hot plate and place the narrow strip on the slide to dry (Fig. I-IB)]. Stain the slides with toluidineblue (Fig. 1-2A) and pyronine (Fig. 1-2B) at low pH by placing a drop of the stain on the surface of the slides and heating until the drop begins to dry from the edges. After washing, the stain is separated by 95% ethanol [Dye preparation: 0.1 g of toluidine blue, 0.05 g of pyronine, and 0.1 g of borax (sodium tetraborate) dissolved in 60 ml of distilled water; the solution should be filtered periodically]. After drying on a hot plate, the slides are sealed with DPX [distrene-plasticizer-xylene] [5 narrow strips of 10 slices each are placed on a slide and sealed with a 50 mm x 22 mm coverslip (Fig. 1-1C)]. Of course, care should be taken to seal the slides with the sectioned side facing up]. Common Problems in the end The primary terminals of the myofibrils in the cat's thin, short, cylindrical muscle occupy the middle part of the myofibrils, which are approximately 350 mm long, and approximately 50 longitudinal serial sections of 1um thickness are required for a complete view of the myofibrils. It is generally accepted that the primary terminus of the myofibers consists of a system of peripheral terminal branches emanating from a single class Ia primary afferent fiber and preterminal branches of myelinated or unmyelinated fibers, which are often distributed among the intramuscular fibers of the shuttle. The endocardium usually consists of six muscle fibers of three different types. Figure 1-3 shows photographs taken with a 100× achromatic oil microscope (NA = 1.25); most of the structures less than 0.5/um in size have been lost to lysis. Each field of view contains a section of the muscle shuttle that is more than 100 beers long. The most prominent structure seen in the photographs is the middle portion of an intraclavicular muscle fiber, the nuclear pouch fiber. : In the primary end zone, the myotome of the intraclavicular fiber is almost entirely replaced by aggregated nuclei (n in Fig. 1-3C). The projections on the surface of the fibers are sensory terminals (t in Fig. 1-3F). The preterminal branching portions of the sensory fibers, both medullated (mpt in Fig. 1-3B) and unmedullated (pt in Fig. 1-3E), are seen in these sections. The dark structures within the terminals were mainly mitochondria. Several accessory, fibroblast-like cells surround the pouch fibers and form a sheath-Figure 1-3 G is an outline of a pouch fiber with sensory endings attached to its surface based on the remodeling of adjacent serial sections. Banks published a three-dimensional remodeling of intact endings in 1986, and in 1997 Banks et al. applied a similar serial section analysis to multiple coding sites in primary endings and to the pectoral controller (pacemaker). In 1997, Banks et al. applied similar sequential sectioning analysis to the multiple coding sites of primary endpoints and to the combined tissue-physiology of the interaction with the pacemaker. For more product details, please visit Aladdin Scientific website.
Sodium dimethylarsenate buffer Glutaraldehyde Osmium tetroxide Ethanol Ethylene oxide Toluidine blue Pyronin
Slide
Dehydration and Embedding
Sectioning and staining
