Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

A comparison of aldehyde fuchsin and alcian blue staining of neurosecretory material in Oncopeltus fasciatus

The dynamics of the "A" cells of the parsintercerebralis of Oncopeltus fasciatus over the first eight days of adult life was studied by microspectrophotometry of sections stained either with aldehyde fuchsin or alcian blue 8 GX. The data show that the two stains differ in their selectivity as they record different events in the history of the cells. A hypothesis is proposed that the aldehyde fuchsin is more sensitive to the presence of a "carrier" protein in the cell, whereas alcian blue 8 GX is more sensitive to the presence of the "active principle" in the cell.     

Detection of pinocytic activity using selective staining with phosphotungstic acid

Summary Post-staining with phosphotungstic acid has been investigated using thin sections of isolated tomato fruit protoplast material. Certain areas of the plasmalemma and some cytoplasmic vesicles were found to stain intensely, and this selectivity of staining has been shown to be associated with pinocytic activity.This work was supported by a special grant from the S.R.C. One of us (MAM) acknowledges the award of an S.R.C. Research Studentship. This work is part of a thesis approved for the degree of Ph.D. in the University of Nottingham (MAM).     

An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels

Partially deglycosylated chondroitin sulfate proteoglycan (CSPG) or peptide fragments obtained from CSPG are not readily detectable in gels by staining with Alcian blue 8GX or ammoniacal silver using the technique of Oakley et al. (B. Oakley, D. Kirsh, and N. Morris (1980) Anal. Biochem. 105, 361). Sequencial staining with both reagents allows visualization of intact CSPG or peptides derived from proteoglycans in polyacrylamide gels at protein concentrations as low as 2 ng/mm2, or glucuronic acid and galactosamine concentrations of 1 ng/mm2 or less. This method is significantly more sensitive and has broader applicability than that described by H. Min and M. Cowman (1986) Anal. Biochem. 155, 275) for staining glycosaminoglycan fragments in polyacrylamide gels.     

A method of processing tissue sections for staining with cu-promeronic blue and other dyes, using CEC techniques, for light and electron microscopy

The application to connective tissues etc. of new histochemical and ultrastructural methods involving consecutive treatment with dye-salt baths, specific enzymes, antibodies and decalcifying fluids, is best carried out on sections on glass slides. This ensures uniform access of reagents to substrates and allows the accurate monitoring of the results, as well as exploiting the strengths of the newer reagents (e.g. Cupromeronic Blue) in that they are highly coloured as well as electron dense. However, very high losses of connective tissue sections usually occur in the various solutions, to the extent that many important investigations are rendered almost impossible because of the high cost of the reagents. We describe a technique which we use routinely, that gives very high recovery of sections of "difficult" tissues from multi-stage enzyme and stain treatments, for light microscopy followed by plastic embedding of the coloured sections for electron microscopy.     

Ultrastructure of Kurloff body proteoglycans after high pressure freezing, cryosubstitution and postembedding staining with cuprolinic blue

Very high pressure freezing and cryosubstitutlon of Kurloffcells preserves the ultrastructural morphology of Kurloff bodies,particularly the myelin figures, as shown by embedding in epoxyresin and conventional postembedding staining. It also preservesthe Kurloff body proteoglycans as more expanded spindle-likeshapes than does fixation with formaldehyde at atmospheric pressure.But., proteoglycans were not discernible in the Kurloff bodymatrix on either unstained or conventionally stained thin sections.The Kurloff body skeleton of proteoglycans in their native expandedshape was stained with the electron-dense cationic ministaincuprolinic blue, using thin sections embedded in LR white. Themean equatorial diameter of the spindles was 20–30 nm,while the collapsed filaments produced by aldehyde fixationwere about 10–15 nm wide. The spindles were often about200–300 nm long but could be much longer, depending onthe plane of the section. Thus, high pressure freezing, freezesubstitution, embedding in LR white, and staining with cationicdyes such as phthalocyanins seems to be a convenient way ofvisualizing intracellular proteoglycans that are well preservedand in very much like their native expanded state. cuprolinic blue high pressure freezing Kurloff cell proteoglycans ultrastructure     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Anisotropic staining of apurinic acid with toluidine blue

Summary Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Demonstration of PAS-positive material in electron microscopy with lead staining

Summary 1. Two types of lead staining for electron microscopy, with different staining mechanisms, are described.2. The first type of staining, leading to an increase of the contrast already available, is referred to as intensifying staining.3. The second type of staining leads to the appearance of lead precipitates at several sites where PAS-positive material can be expected. This type of staining is therefore referred to as PAS-like staining.4. Preliminary hypotheses for the mechanisms of these different stainings are given.With 10 Figures in the Text     

A method for staining actin-containing structures in thick plastic sections for medium voltage electron microscopy

A staining procedure has been developed for imaging actin-containing structures in thick plastic sections in the electron microscope. The stress fibres of a fibroblastic cell line were used as a model system, and were first characterized immunocytochemically. After fixation of cells in formaldehyde, mordanting in a solution of gadolinium chloride allows stress fibres to be stained for light microscopy with haematoxylin. A brief exposure to a solution of ammonium paramolybdate renders haematoxylin-stained structures sufficiently electron-dense to be imaged in 1 micron thick plastic sections in a JEOL 200CX electron microscope, operating at 200 kV, and possibly in conventional instruments operating at 100 kV, particularly if equipped with a lanthanum hexaboride source.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.