The Effect of Incomplete Fixation of Elastin on the Appearance of Pulmonary Alveoli

1982 ◽  
Vol 104 (1) ◽  
pp. 68-71 ◽  
Author(s):  
S. S. Sobin ◽  
Y.-C. B. Fung ◽  
H. M. Tremer
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Nonpolar Solvent ◽  
Pulmonary Alveoli ◽  
Histological Sections ◽  
Osmium Tetroxide Solution ◽  
Quick Freezing ◽  
The Difference

Pulmonary alveolar geometry in histological sections prepared by quick-freezing the lung, with freeze-substitution of ice, fixation of the dry lung with osmium tetroxide in nonaqueous and nonpolar solvent and celloidin embedding is remarkably different from the geometry seen in preparations made by instilling aqueous aldehyde or osmium tetroxide solution in the airways. The alveolar walls in the quick-frozen lung are linear; those in the others are wrinkled. The difference was demonstrated to be due to incomplete fixation of elastin.

Destruction of Actin Filaments by Osmium Tetroxide

1977 ◽  
Vol 35 ◽  
pp. 466-467
Author(s):  
P. Maupin-Szamier ◽  
T. D. Pollard
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Osmium Tetroxide ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Sodium Phosphate Buffer ◽  
Transmission Electron ◽  
The Difference ◽  
Rates Of Decay ◽  
Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits

1990 ◽  
Vol 48 (3) ◽  
pp. 42-43
Author(s):  
Marie-Thérèse Nicolas
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Acrylic Resin ◽  
Expected Improvement ◽  
List Type ◽  
Chemical Fixation ◽  
Freeze Fixation ◽  
Liquid Chemical ◽  
Luciferase Enzyme ◽  
Lower Background

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

scholarly journals Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

2022 ◽  
Author(s):  
Martin Schauflinger ◽  
Tim Bergner ◽  
Gregor Neusser ◽  
Christine Kranz ◽  
Clarissa Read
Keyword(s):  
High Pressure ◽  
Potassium Permanganate ◽  
Lipid Membranes ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Block Face ◽  
Critical Aspects

AbstractHigh-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Field Emission Scanning Electron Microscopy Of Mouse Cerebellar Synaptic Contacts

2000 ◽  
Vol 6 (S2) ◽  
pp. 844-845
Author(s):  
O.J. Castejón ◽  
R. P. Apkarian ◽  
H. V. Castejón
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Field Emission ◽  
Cerebellar Cortex ◽  
Osmium Tetroxide ◽  
Phosphate Buffer Solution ◽  
Buffer Solution ◽  
Solution Ph ◽  
Osmium Tetroxide Solution ◽  
Scanning Electron

Samples of albino mice cerebellar cortex were processed by the cryofracture method for scanning electron microscopy and examined with the field emission scanning electron microscope (FESEM). Albino mouse cerebellar cortex was excised, cut into 1-2 mm slices and inmersed in 4% glutaraldehyde in O. l M phosphate buffer solution, pH 7.4, for 24h at 4°C; and postfixed for 1 h in a similarly buffered 1% osmium tetroxide solution. Specimens were dehydrated in a graded serie of ethanol (30, 50, 70, 80, 90 2x100%) prior to wrapping individual tissue pieces in preformed absolute ethanol filled parafilm cryofracture packets. Rapid freezing of packets was performed by plunging into LN2. First, the packet was transferred from the LN2 storage vessel with LNT chilled forceps in order to avoid themial damage. Secondly, the cooled fracture blade was removed from the LN2, the packet was orientated under the blade, and immediately struck with a heavy tool.

THE SECRETORY PATHWAY IN PLATELETS STUDIED BY CRYO-FIXATION

1987 ◽  
Author(s):  
E morqenstern ◽  
H Patscheke
Keyword(s):  
Osmium Tetroxide ◽  
Secretory Pathway ◽  
Freeze Substitution ◽  
Human Platelets ◽  
Dense Matrix ◽  
Compound Exocytosis ◽  
Ultrathin Sections ◽  
Granule Secretion ◽  
Large Compound ◽  
Electron Dense Matrix

It is widely held, that the constituents packed in the a -granules are released by stimulated platelets via the surface connected system (SCS). By means of the fast-freezing and freeze substitution technique (which allow the investigation of membrane fusion) we found a secretory pathway in platelets (compound exocytosis) without an involvement of the SCS during the release of a-granules. To study the process of a-granule secretion human platelets concentrated in citrated blood plasm were stimulated with thrombin or collagen. 20 - 120 seconds after stimulation the platelets were rapidly frozen with a metal-mirror attachment to the KF 80 cryofixation unit (REICHERT-JUNG). Using plastic spacers droplets of the PRP were slammed against a copper block at 80 K at a rate of 0.2 m/sec. After cryofixation the specimens were transferred (in liquid nitrogen) into a Cs-auto cryosubstitution unit (REICHERT-JUNG). Cryosubstitution was programmed for 48h at 193 K in acetone with 4% osmium tetroxide. The temperature went automatically up to room temperature at a rate of 10 K/h. The specimens were embedded in araldite. The analysis of serial ultrathin sections of platelets in different phases of exocytosis revealed the following. a -granules in apposition showed different stages of swelling and dispersal of their electron dense matrix. Membrane appositions were also found between a -granules. The contraction of a sphere of microfilaments and microtubules during stimulation seemed to support this process. On the other hand this internal contraction prevented most of the a-granules from contacting with the plasmalemma. We observed fusion between swollen -granules in apposition and the plasmalemma and swollen and unswollen a -granules. Thus, large compound granules were formed frequently before fusion of the secretory organelles with the plasmalemma took place. These observations suggested that a -granules in stimulated platelets performed a compound exocytosis after swelling. The process seemed to start with the apposition of a -granule membranes to the plasmalemma. It cannot yet be answered whether the swelling of the granules is due to an osmotically driven influx of water or due to an influx after microfusion.Supported by DFG, Grant Mo 124/2-4

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