An efficient staining method for ultrathin sections.

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

Antikeratin antibody staining on ultrathin sections of epidermal cells prepared by low-denaturation embedding

Direct immunological identification of cellular components has not been possible in tissues prepared for electron microscopy by conventional methods. This may be attributed, in part, to the relatively harsh reagents employed. Using an approach to preparation of biological specimens for transmission electron microscopy that aims for minimal perturbation of native protein conformation, we have obtained specimens that may be stained with antibodies. Recent investigations using these methods have revealed new information regarding the organization of epidermal cells.     

The loss of enzyme reaction products from ultrathin sections during the staining for electron microscopy

Summary The effects of heavy metal salt staining procedures on the reaction products obtained in the demonstration of arylsulphatase and of acid phosphatase were studied.Lead citrate staining at pH 12 was found to cause a very marked dissolution of barium sulphate and a moderate dissolution of lead sulphate. The staining with uranyl acetate was found to dissolve moderately both barium and lead sulphate.Neither lead citrate nor uranyl acetate staining had any remarkable effect on lead phosphate.The mechanism of the dissolution and the possibilities to avoid it were discussed.     

Use of Hydrogen Peroxide to Accelerate Staining of Ultrathin Spurr Sections

Pretreatment of ultrathin Spurr sections of glutaraldehyde-osmium fixed tissue with hydrogen peroxide significantly reduces the time required to stain with ethanolic uranyl acetate and lead citrate for electron microscopy. Micrographs compare contrast obtained by this method with that obtained using conventional staining times. Reasons for the facilitating action of hydrogen peroxide are suggested.     

Intracellular localization of certain membrane glycoproteins in mouse T-lymphoma cells using immunoferritin staining of ultrathin frozen sections

Comparative studies on the cellular morphology of culturd mouse T-lymphoma cells (with particular emphasis on organelles and membrane-associated materials) were conducted using both frozen thin sections and epon thin sections. Due to the fact that the frozen thin sectioning technique allows antigenicity to be retained and also permits good accessibility of the external macromolecular reagents to the interior of the cell, we have been able to explore the intracellular localization of some membrane glycoproteins such as Con A-binding sites and viral membrane glycoprotein, gp 69/71. Our data indicate that most of the membranous cellular structures (e.g., rough endoplasmic reticulum, vesicles, Golgi and nuclear envelope) contain the Con A-specific sugars, mannose, and glucose. In addition, we have found that intracellular gp 69/71 molecules exist in an aggregated form at the terminal region of cisternae of rough endoplasmic reticulum and in vesicles of two size ranges (0.1 to 0.15 microns and 0.3 to 0.4 microns) as well as in the cytoplasm close to the plasma membrane. These findings have not only confirmed some of the previous biochemical data but have also provided new information concerning the biochemical nature of intracellular membrane components and the possible biosynthetic fate of membrane precursor molecules.     

Intensive drying of grids after lead citrate staining lowers precipitate contamination on the surface of ultrathin sections

Two techniques are proposed to minimize precipitate contamination of ultrathin sections after lead citrate staining. According to the first one, stained and washed grids are placed, sections downward, on a stack of filter papers. The second one involves a consecutive washing of grids with distilled water, 96% ethanol, and n-hexane. Both techniques are equally efficient; the former being simpler in processing, while the latter is superior in the reproducibility of results.     

Use of hydrogen peroxide-egg albumin to eliminate nonspecific staining in immunoperoxidase techniques.

The application of hydrogen peroxide and egg albumin to paraffin sections before immunostaining prevents nonspecific staining by immunoperoxidase techniques. This method is more effective than pretreating secions with normal sera, or using either egg albumin or hydrogen peroxide separately, or using diluted antisera with prolonged incuabations in the staining procedure.     

Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts

Infections by the protozoan parasite Cryptosporidium parvum are routinely diagnosed by modified Ziehl-Neelsen (acid-fast) staining of faecal preparations despite the counterstaining and ghost-like appearance of some oocysts. Quantitative studies demonstrated that only a small percentage of oocysts excreted by naturally infected newborn calves displayed acid-fast characteristics, but that percentage increased when the time between excretion and sample staining was increased. The treatment of faecal samples with hydrogen peroxide (10 min, 5 vol. final concentration) caused all oocysts to become acid-fast, with up to 40-fold increases in test sensitivity in samples treated and stained within 3 h of excretion. Flow-cytometry analysis of hydrogen peroxide-treated oocysts also demonstrated increased labelling of oocysts by a commercial monoclonal antibody preparation commonly used for diagnosis.     

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.     

Use of hydrogen peroxide for closing disulfide bridges in peptides

The use of hydrogen peroxide for the formation of disulfide bridges was studied in 15 peptides of various lengths and structures. The oxidation of peptide thiols by hydrogen peroxide was shown to proceed under mild conditions without noticeable side reactions of Trp, Tyr, and Met residues. Yields of the corresponding cyclic disulfides were high and mostly exceeded those obtained with other oxidative agents, in particular, iodine. It was established that the use of hydrogen peroxide in organic medium also provided sufficiently high yields when large-scale syntheses of oxytocin and octreotide (up to 10 g) were carried out. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.