Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase.

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.     

Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described. Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min. The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining.     

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

The in situ staining of muscles during development with the cholinesterase technique

Developing muscles from forelegs of 11- to 18-day-old mouse embryos were stained in situ for cholinesterase with the copper-ferrocyanide technique. The skin of the legs represents a diffusion barrier for the incubation medium. Therefore, in older embryos the skin was mechanically removed after trypsin digestion. In younger embryos the skin remained on the forelegs after trypsin treatment. With this technique it is possible to follow the establishment of the muscular pattern in the legs.     

An acetylcholinesterase method for in toto staining of peripheral nerves.

Stomach, small intestine, uterus, urinary bladder, vagina, mesentery, mesometrium and joint capsule of rats, gall bladder, cystic duct and bile duct of dogs and uteri of young children are stained in toto. Procedure: Tissue is perfused with saline containing hyaluronidase, then pinned on a flat layer of Paraplast and fixed for 24 hr in cold sucrose formol solution. Stomach, urinary bladder and gall bladder are also fixed in toto. Rinse for 2 days in cold 0.22 M sucrose in a sodium cacodylate buffer pH 7.2. Incubate in medium consisting of 60 mM acetate-buffer pH 5.0 or pH 5.6 (for human material only), 2 mM acetylthiocholine iodide, 15 mM Na citrate, 3 mM Cu sulphate, 0.5 mM K3Fe(CN)6, 5 times 10-4 M iso-OMPA, 1% Triton X 100 at 37C. Rinse in doubly distilled water. Dehydrate in glycerine/water mixtures of increasing glycerine content. Store in glycerine or delaminate under dissecting microscope. Delaminated specimens are mounted on gelatinized object glasses, cleared in xylene and coverslipped with Malinol. Specimens stored in glycerine can be studied microscopically. Stained specimens can also be embedded in Paraplast and sections can be studied after counterstaining.     

基于MTT染色法对葡萄生单轴霉孢子囊存活力的检测

本文利用MTT染色法和点接生物测定法对不同温度干燥处理36h后的葡萄生单轴霉孢子囊存活力和致病力进行了检测,并应用单因素试验和正交试验对其孢子囊进行了MTT染色条件的优化。结果表明：葡萄生单轴霉孢子囊MTT染色的最佳条件为温度36℃、MTT浓度 0.05%、染色48h,孢子囊染色率可达83.0%。20℃恒温干燥处理36h显著提高了孢子囊存活力,葡萄生单轴霉孢子囊的蓝色染色率为79.3%,叶片点接发病率为98.9%,显著高于对照的蓝色染色率52.0%和发病率62.7%。MTT染色得到的孢子囊蓝色染色率与点接生物测定法得出的发病率存在很好的线性关系y=1.276 1x-1.939 1,R²=0.996 1,孢子囊的蓝色染色率与叶片点接发病率呈正相关。本研究表明MTT染色法可以用于葡萄生单轴霉孢子囊存活力的快速和准确检测。     

Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.     

Expression of cholinesterase in the Schwann cells of peripheral nerves during development

In peripheral nerves of mouse embryos Schwann cells exhibit a high activity of unspecific cholinesterase. At first (day 12 of embryonic development) this enzyme occurs in the nuclear envelope and in the granular endoplasmic reticulum. Thus, it is possible to differentiate between Schwann cells and fibroblasts which lack cholinesterase. Later on (day 16) the cholinesterase has shifted to the cell membrane of the Schwann cells. However, only that part of the plasmalemma which encircles single axons and the mesaxons exhibits an irregular deposition of the reaction end product. In newborns the first loops of the just formed myelin sheath are still stained. With maturation of the myelin sheath the enzyme activity disappears. The functional role of cholinesterase is unclear. Possible roles are discussed. The expression of cholinesterase in Schwann cells depends on the integrity of the axons. After a few hours, the cultivation of amputated limbs results in a reduction of the enzyme activity. After 1 day in culture cholinesterase disappears totally.     

A study of the peripheral quaternary ligand binding site of cholinesterase with the use of ethidium bromide

The peripheral binding site of horse serum cholinesterase (EC 3.1.1.7) for quaternary ligands was investigated by fluorescent probing with the use of ethidium bromide. Spectral evidence for the participation of the tryptophan indole group of the peripheral site of horse serum cholinesterase in the formation of a cholinesterase complex with ethidium bromide is presented. The mechanism of cholinesterase effect on ethidium bromide fluorescence is proposed.     

The desmosome: Fine structural studies with freeze-fracture replication and tannic acid staining of sectioned epidermis

Desmosomes of larval and post-metamorphic newt epidermis have been studied by freeze-fracture replication both with and without prior glutaraldehyde fixation. Characteristic particles of a diameter (70-130 A) similar to that of typical membrane associated particles are found clustered on the exposed internal faces of adherent desmosomal membranes. They remain attached to the B-face in unfixed material, but occupy the desmosomal A-face after fixation. Membrane associated particles of nondesmosomal surfaces are found predominantly on the A-face in both fixed and unfixed epidermis. Suitably oriented replicas of unfixed desmosomes reveal profiles of apparent fine filaments extending from the region of tonofilament loops through the desmosomal plaque to traverse the cytoplasmic leaflet of the plasmalemma. They can be traced onto the B-face. Their position correlates to fine linear profiles noted in tannic acid/glutaraldehyde-fixed and sectioned desmosomes. The possibility that these represent a mechanism for anchorage of tonofilaments to the plaque and to the membrane is discussed. These and other fine structural features are compared and contrasted to the properties of hemidesmosomes described in the preceding report.