Fixatives osmium tetroxide

These bands were a matter of discussion for a long time until in 1954, for the first time, Sjostrand and Andersson used electron microscopy to investigate intercalated disks in ultrathin osmium tetroxide-fixed sections of the mouse heart revealing that the disks were indeed transverse cell boundaries. Subsequently, several investigators reproduced their finding [Lindner, 1957 Moore... 

Figure 10.8 Milk lipid globule membranes released by churning of washed globules and collected by ultracentrifugation retain densely staining coal material along one face of the bilayer membrane. As seen in this electron micrograph of glutaraldehyde and osmium tetroxide-fixed material, the...

FIGURE 7.7. Thin sections of glutaraidehyde- and osmium tetroxide-fixed human biopsy heart tissue. 

FIGURE 18.8 (a) Osmium tetroxide-fixed stratum corneum. (i) Control tissue no treatment and incubated at 44% RH. Note electron dense comeodesmosomes are fully... 

Osmium tetroxide fixative 2% (w/v) OSO4 in 0 12M sodium phosphate, pH 7 2 (Take care poisonous ). 

Aspirate the wash buffer and fix approx 1 mm pieces of each pellet in 1 mL of osmium tetroxide fixative for 60 min at 20 C This procedure will further harden the pellets. 

The size of each fixed pellet should not significantly exceed 1 mm, otherwise the osmium tetroxide fixative (see Section 3.3., step 8) will not be able to penetrate effectively to the center of the pellet. 

One of the main uses of osmium tetroxide is as a biological staining agent for microscopic ceU and tissue studies. Osmium tetroxide is unique in that it both fixes and stains biological material. 

The Smith technique consisted of fixing the smear in osmium tetroxide vapor, immersion in HCI, mordanting in dilute formaldehyde, and staining with aqueous basic fuchsin. The method was said to possess certain advantages over the procedure of Robinow. 

Harvested samples should be immediately prepared for the SEM by fixation in a 0.5% osmium tetroxide solution in the dark for 1 h. If the sample is collected in the field, store in buffered 3% glutaraldehyde solution (pH 7.2) until samples can be fixed. 

Matus AI. Ultrastructure of the superior cervical ganglion fixed with zinc iodide and osmium tetroxide. Brain Res 1970 17 195-203. 

Epon 812 (Polybed) Formvar Glutaraldehyde Ilford L-4 emulsion with appropriate safelight Osmium tetroxide Phosphate Dibasic Monobasic Propylene oxide Sodium thiosulfate fixing solution... 

Tissue processing tissue specimen (0.5 1.0 mm3) are fixed in 4% buffered formalin for 30 60 min, post-fixed in 1% osmium tetroxide in cacodylate or phosphate buffer, pH 7.2 7.4, stained en bloc for 30 min with 2% aqueous uranyl acetate, then dehydrated in ethanol and embedded in epoxy or acrylic resin. 

Lesion in bovine dentin with tubules protruding from degraded intertubular matrix (left degraded matrix right intact matrix). Demineralization in 0.1 M acetic acid pH 4.0, with subsequent exposure to bacterial collagenase. Fixed and demineralized with glutar-dialdehyde-acetic acid, post-fixed with osmium tetroxide ultrathin sections stained with uranyl acetate - lead citrate. 

If a slice of fresh (frozen) tissue is examined directly, little is seen because most of the atoms found in cells are of low atomic mass and scatter electrons weakly and uniformly. Therefore, thin sections must be "stained with atoms of high atomic mass, e.g., by treatment with potassium permanganate or osmium tetroxide. Tissues must also be "fixed" to prevent disruption of cell structures during the process of... 

After glutaraldehyde fixation (Section 3.1 1 2., step 8), fix the cells further with 1% osmium tetroxide, saturated uranyl acetate, dehydrate in an ascending series of ethanol (70, 90, 100%), and embed in epoxy resin. Ultrathin sections of the block, stained with 1% methanolic uranyl acetate and lead citrate, will reveal immunolabeling on the outer surface of the cells... 

The immunoreplica technique (14) is used when it is necessary to detect antigenic sites on the plasma membrane of cultured cells. The cells are cultured on coverslips, and are fixed as described above depending on the antibody in question, and immunolabeled in situ as described in Section 3.1.1.2., steps 3-9. After immunolabeling (Section 3.1.1.2., step 9), they are further fixed with 1% osmium tetroxide and are dehydrated in a graded series of ethanol (70, 90, 100%), critically point-dried, and replicated with a layer of carbon and platinum, The replicas are cleaned with sodium hypochlorite and chronic acid before examination with the transmission electron microscope. Large areas of the replicated plasma membrane remain intact for observation. Colloidal gold probes are probably the only probes of sufficient density that can be detected on these surfaces. 

For electron microscopical analyses, the fixative was 2.5% glutaraldehyde followed by 1% osmium tetroxide for 1 h at 4°C, and embedding in an epon-araldite mixture (see below). 

For conventional electron microscopy, processing was performed as described in Tsukada et al. (2001) and Yamashima et al. (2003). Small specimens of hippocampus were fixed with 2.5% glutaraldehyde for 2 h and subsequently with 1% osmium tetroxide in TBS for 1 h at 4°C, dehydrated in ascending ethanol series, incubated in propylene oxide (2 x 15 min) and finally embedded in epon-araldite mixture. Usually, between four and eight semithin (1 pm) sections were stained with tolui-dine blue for the light microscopic observation. After trimming upon observation of toluidine blue staining, the ultrathin sections were stained with uranyl acetate (10 min) and lead citrate (5 min) for the electron microscopic observation (JEM H-600, Hitachi, Tokyo). 

After primary fixation the algal and root samples were rinsed in 0.1 M PIPES buffer, pH 7.0, then twice in 0.1 M sodium cacodylate buffer, pH 6.8, and post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 6.8, overnight at 4°C. The osmium solution prepared for the root samples was added with 0.7% potassium ferricyanide in order to improve osmium penetration in the root tissues. This was particularly necessary for the 2 h control roots and roots from RO-treated seed, due to their relatively low water content. The replacement of PIPES buffer with cacodylate buffer before osmication was necessary, as PIPES reacts with osmium, producing a dark precipitate. 

Specialty fixatives, such as Osmium tetroxide, which is used primarily in electron micrography and acetone, is used in fixation of frozen sections. Others used for research purposes on specific tissue, organs or even whole organisms are not discussed in this publication. 

Figure 4.3. Electron micrograph showing a cytoplasmic lipid droplet (cld) and numerous microlipid droplets, some of which are denoted by arrows. An apparent fusion figure between the cld and a microlipid droplet is denoted by the arrow on the right. The specimen was fixed simultaneously with glutaraldehyde and osmium tetroxide. Bar = 0.5 p.m.

After the last experiments, the rats are euthanized by injection of an overdose of pentobarbital and then perfused intracardially with a phosphate-buffered 2.0% paraformadehyde - 2.5% glutaraldehyde fixative. Methyl green solution was injected to confirm the location of the catheter after the perfusion. The spinal cord and nerve roots were dissected out and immersed in the same fixative for 4 h. Two specimens (10 mm rostral and caudal to the conus medullaris from each rat were postfixed with cacodylate-buffered 1 % osmium tetroxide dehydrated in a series of graded alcohol solutions, and embedded in epoxy resin. From the embedded tissue, 1-pm transverse sections were obtained and stained with toluidine blue dyes. Sections obtained from 10 mm rostral to the conus (caudal spinal cord) were used for qualitative evaluation. Quantitative analysis of nerve injury was performed using the sections obtained form 10 mm caudal to the conus. Each fascicle present in the cross section was assigned to an injury score 0 to 3. The injury score for each cross section was then calculated as the average score of all fascicles present in the cross section. 

Fig. 2. Electron micrograph of vanadocytes from Ascidia nigra. Cells were fixed in 2.5% glutaral-dehyde in 0.15 M HC1, followed by 1% osmium tetroxide, pH 7.6. The sections were stained with uranyl acetate N = nucleus, PM = plasmalemma, V = vacuoles (very electron dense presumably because of reduction of osmium tetroxide by V(III).)...

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