Ultrastructural experiments on cells
Ultrastructural experiments on cells
Although electron microscopy cannot observe living cells and tissues, the advent of electron microscopy still revolutionized the development of microscopy. Since it is not possible to directly compare the structural phase with that of living cells, the goodness of ultrastructural fixation can only be evaluated subjectively, but physical fixation of living tissues by rapid freezing is completely feasible (see Vema1983 for details), which provides an objective criteria for evaluating the effectiveness of chemical fixation.
Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Operation method
Synaptic experiments in the cerebellar cortex
Materials and Instruments
Cerebellar cortex of adult rats Cerebellum removed from rats anesthetized by intraperitoneal injection of sodium pentobarbital Move I. Fixed 1. Perfusion with Kamovsky fixative. Kamovsky fixative is formulated as follows (in 100 ml volume of fixative): Liquid A: 2 g paraformaldehyde is dissolved in 40 ml of water at 60°C. 2 to 6 drops of 1 mol/L NaOH are added slowly until the solution becomes clear. The two liquids were stored in 4L before use, and then mixed into 100 ml of fixative when used. The detailed steps vary between perfusion methods, and the perfusion method used here is both simple and reliable in that it minimizes the time between initiation of anesthesia and effective fixation. A peristaltic pressurized pump (Watson-Marlow MHRE200) powers the perfusion (hydrostatic pressure has also been used by many authors), and the fixative is introduced immediately after intubation. The flow rate of the fluid was kept relatively low initially and was gradually increased for a short period of time after the signs of immobilization (limb and tail extension). The fixation process lasts approximately 10 min and 500 ml of fixative is required to fixate one adult rat. There is no need for additional monitoring of pressure during perfusion. The cannula is made from a 21 G hypodermic needle, bent at the midpoint and the tip smoothed. A drop of epoxy resin was placed on one side of the needle tip and then applied around the tip to facilitate insertion of the catheter into the ascending aorta through an incision in the left ventricular cusp. After cannulation, the cannula and heart were clamped together with an arterial clamp and placed in the proper position. The peristaltic pressurization pump should be kept running continuously at a low speed during the procedure and cannulation to prevent air bubbles from entering the vessel. As soon as the cannula is secured in the proper position, the right atrium is clipped while the pump rotation is accelerated and fixation fluid is introduced to begin fixation. 2. After perfusion is complete, the brain is removed and placed in fresh fixative. Tissue blocks or slices should be cut thin enough (no more than 1 mm) to facilitate osmium tetroxide penetration. Osmium tetroxide post-fixation, dehydration, and embedding procedures are the same as those described in Example 1 above. 1. Sections are 1 um thick, stained with toluidine blue and Pyronin for initial observation and adjustment, as in Example 1. 2. Sections about 70~90 nm thick (silver-white-pale golden-yellow in color) were cut with an ultrathin slicer, collected on a copper mesh coated with polyvinyl alcohol, dried and stained with lead citrate and uranyl acetate. Common Problems in the end Several different types of synapses have been described within the cerebellar cortex, most of which belong to axial-dendritic synapses. We will only briefly mention three examples here: one from the molecular layer and the other two from synaptic glomeruli within the granular layer. As far as synapses are concerned, the synaptic connections within the molecular layer are mainly formed by the parallel fibers of granule cells with the dendritic spines of Purkinje cells. A horizontal section of the outermost layer of the molecular layer containing transected parallel fibers (pf) is shown in Figure 1-4A. Several synapse(s) formed by parallel fibers with dendritic spines are visible; note that these synapses often form close connections with glial cell protrusions, whereas the parallel fibers (axons of granule cells) are clustered together and lack the individual myelin sheaths formed by glial cells. Figure 1-4B is a magnified image of a synapse of the same type, a Grayl-type synapse. Of particular note is the thickening of the postsynaptic membrane and the presence of a thin layer of extracellular matrix between the presynaptic and postsynaptic membranes. The presynaptic vesicle is round (rv). Figure 1-4C shows a synaptic vesicle within the granular layer, which is a complex structure formed by a central rose-petal-shaped terminal clasp of mossy fibers (mf) surrounded by numerous dendrites (gcd) of granule cells and axons of Golgi cells. Both mossy fibers and Golgi cell axons form axial-dendritic synapses with granule cell dendrites, but they belong to the Gray1 and Gray2 types of synapses, respectively. Figs. 1-4D Large size shows the synapses between Gdgi cells and granule cells. The thickening of the postsynaptic membrane is much less obvious than that of Grayl-type synapses, and there is no obvious extracellular matrix between the presynaptic and postsynaptic membranes. Many presynaptic vesicles are flat vesicles. Previous studies have revealed that parallel and mossy fibers are excitatory, whereas Golgi cells are inhibitory. For more product details, please visit Aladdin Scientific website.
Kamovsky's fixative, paraformaldehyde, glutaraldehyde, sodium dimethylarsenate.
Liquid B: 25% glutaraldehyde 10 ml mixed with 0.2md/L sodium dimethylarsenate 50 ml, pH 7.3.
