Staining of free sulfhydryl groups of proteins after separation by isoelectric focusing of Bio-Rad standards and lens crystallins.

In this study, we propose a new staining method for free sulfhydryl groups of proteins after having separated native samples by thin-layer isoelectric focusing (IEF) in absence of detergents. A comparison was made between proteins stained purple for free sulfhydryl groups (SH) and proteins stained blue by Coomassie blue (CB). A stainability factor, F = %SH/%CB, was calculated for each protein. The Bio-Rad IEF standards containing seven marker proteins for pH scale determination were stained purple, in the same way as they were designed for CB staining. To prove the validity of the currently proposed staining method for a defined protein system such as the eye lens crystallins, these proteins were also stained after IEF as described above. The factor F was calculated for all alpha-, beta-, and gamma-crystallin components that stained in both methods. We discovered that alpha-crystallins contained comparatively high amounts of free SH groups, while some beta- and gamma-crystallin components also contained considerable amounts of free SH groups. The SH staining procedure with 2,2'-dihydroxy-6,6'-dinaphthyl disulfide applied after IEF appeared to be useful, specific, and reproducible.     

Abundance of protein-bound sulfhydryl and bisulfide groups at chromosomal nucleolus organizing regions

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.     

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicer's hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiéry's periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.     

True enamel matrix of the newt,Triturus pyrrhogaster,contains no sulfated glycoconjugates

Summary The ultrastructural distibution and histochemical properties of sulfated glycoconjugates were investigated in the developing enamel of the adult newt, Triturus pyrrhogaster, by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Development and ultrastructure of the enamel were also studied. After deposition of the mantle dentin matrix to a certain thickness, the first enamel matrix, globular in shape, appeared in juxtaposition to the dental basement membrane and tended to be intermixed with the previously deposited dentin matrix. Subsequently, enamel matrix was deposited outside (ameloblastic side) of the dental basal lamina and formed a true enamel layer. Thus, developing enamel of the newt consists of two layers: (1) an inner layer made up of a dentin-enamel mixed matrix and (2) an outer layer composed of only true enamel matrix. HID-TCH-SP precipitates resulting from the abovementioned studies were found in the mixed matrix and were identified as chondroitin sulfates; in contrast, the true enamel matrix contained no sulfated glycoconjugates.     

Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultraviolet absorbing dyes as histochemical reagents

Summary Three biochemical methods depending on measurement of absorption in the ultraviolet range were successfuly adapted to histochemical techniques to be used in conjunction with ultraviolet microscopy. They included cetyl pyridinium chloride (max. = ca. 260 nm) as a stain for acid mucopolysaccharides; picryl sulfonic acid (max. = ca. 340 nm) as a stain for proteins, particularly amino groups; and fluorescein mercuric acetate (max. = 293 nm) as a stain for protein bound sulfhydryl and disulfide groups. The general possibilities of this approach was discussed.Dedicated to Prof. Dr. Felix Mainx on the occasion of his 70th birthday.The author would like to acknowledge the technical assistance of Dr. Sherill K. Curtis in the development of these investigations.     

Localization of phosphophoryn in rat incisor dentin using immunocytochemical techniques

We studied the distribution of the phosphophoryn present in rat incisors by immunolocalization and histochemical techniques. The polyclonal antibody used reacts with both phosphorylated and de-phosphorylated phosphophoryn. Technical problems encountered in immunostaining and in preparing sections from mineralized dentin were resolved by use of peroxidase-conjugated protein A as the "second antibody" in indirect immunostaining reactions and by surface etching of partially demineralized sections. Staining with anti-rat incisor alpha-phosphophoryn antibody showed light staining over the odontoblasts and proximal odontoblastic processes, no stain over the predentin, dense staining over the intertubular dentin, and no stain over the mantle dentin. In the intertubular dentin the stain intensity was directly related to the distribution of mineral. These findings were directly corroborated by staining with Stains All. The mineralization of dentin and the distribution of phosphophoryn within the dentin may be much less uniform than previously supposed.     

Comparison of tetrachromic VOF stain to other histochemical staining techniques for characterizing stromal soft and hard tissue components

The components of hard tissues including dentin, enamel, cementum, bone and other calcified deposits, and mature and immature collagen pose problems for identification in routine hematoxylin and eosin (H & E) stained sections. Use of combinations of stains can demonstrate the components of hard tissues and soft tissues distinctly. We assessed the efficacy of the Verde Luz-orange G-acid fuchsin (VOF) stain for differentiating hard and soft connective tissues and compared results with other histochemical staining techniques. Eighty tissue sections comprising developing tooth (30), ossifying fibroma (30) and miscellaneous pathologies (20) expected to contain varying types of calcified tissues were stained with H & E, VOF, and Masson's trichrome (MT). In developing tooth, VOF demonstrated better differentiation of hard tissues, while it was comparable to MT for ossifying fibroma and miscellaneous pathologies. The intensity of staining was greater with VOF than with the other stains studied. VOF stains hard tissue components distinctly and gives good contrast with the surrounding connective tissue. VOF is comparable to MT, but has added advantages including single step staining, rapid and easy procedures, and it distinguishes the maturity of the tissues.     

Involvement of sulfhydryl groups of chromosomal proteins in sister chromatid differentiation

The fluorescence of human lymphocyte chromosomes stained with sulfhydryl group-specific fluorochromes is markedly enhanced by a mild near-ultraviolet irradiation pretreatment, indicating breakage of protein disulfide bonds. When metaphase preparations of cells cultured in the presence of BrdU during two cell cycles are irradiated and subsequently stained with the sulfhydryl group-specific fluorescent reagents used in this study, a differential fluorescence of sister chromatids is observed. After staining with the DNA-specific fluorochrome DAPI an opposite pattern of lateral differentiation appears. It can be concluded that the chromatid containing bifilarly BrdU-substituted DNA has a higher content of sulfhydryl groups than the chromatid containing unifilarly BrdU-substituted DNA. This implies a more pronounced effect of breakage of disulfide bonds in the chromatid with the higher degree of BrdU-substitution. BrdU-containing chromosomes pretreated with the mild near-ultraviolet irradiation procedure used by us, do not show any differentiation of sister chromatids after Feulgen staining. Using sulfhydryl group-specific reagents, differential fluorescence of sister chromatids could still be induced by irradiation with near-ultraviolet light after the complete removal of DNA from the chromosomes by incubation with DNase I. Thus, the protein effect of irradiation of BrdU-containing chromosomes takes place independently of what occurs to DNA.Our results indicate that subsequent to the primary alteration of chromatin structure caused by the incorporation of BrdU into DNA, breakage of disulfide bonds of chromosomal proteins might play an important role in bringing about differential staining of sister chromatids, at least for those procedures that use irradiation as a pretreatment or prolonged illumination during microscopic examination.     

THE REACTIVITY AND STAINING OF TISSUE PROTEINS WITH PHOSPHOTUNGSTIC ACID

After aldehyde-fixation, treatment with phosphotungstic acid (PTA) in aqueous acidic medium was shown to produce an intense electron-opaque stain with minimal distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic reticulum, and the Z-band of muscle were densely stained, whereas membranes stood out in negative contrast. Staining of glycogen or lipid was not apparent. Under certain conditions the stain density reflected the concentration of protein based on the quantitative reaction of PTA with the positively charged groups, although the stoichiometry of the reaction between PTA and protein varied with the kind of protein. The staining conditions established should provide a base for the use of the method in quantitative electron microscopy, particularly on thin sections.     

Identification of Mallory bodies with rhodamine B fluorescence and other stains for keratin

Rhodamine B staining in conjunction with fluorescence microscopy is shown to demonstrate Mallory bodies. Mallory body morphology, localization, and distribution in hepatocytes from griseofulvin-fed mice, human hepatoma, and human alcoholics were similar to those observed in the same tissues after conventional staining methods for Mallory bodies. The presence of these inclusions was further confirmed by specific cytochemical localization with indirect immunoperoxidase labeling, horseradish peroxidase labeling, and electron microscopy. Other tinctorial or histochemical procedures previously used for keratin or prekeratin (modified Mallory stain, Kreyberg method, Pauly method for histidine) also stained Mallory bodies for study with white light microscopy but with decreasing sensitivity respectively. Mallory bodies from mouse and human liver both appear to contain a keratin-like moiety. This entity may be simply, rapidly, and permanently stained with rhodamine B, and selectively and reproducibly demonstrated with fluorescence microscopy.     

Iodination-Coupled Tetrazonium and Ferric Ferricya-Nide Reduction Stains for Differentiating Red Blood Cells Containing Fetal or Adult Hemoglobin

Either the iodination-coupled tetrazonium reaction or the ferric ferricyanide reduction procedure can be used to differentiate red blood cells containing fetal hemoglobin (hemoglobin F) from those containing adult hemoglobin (hemoglobin A) in blood smears. Oxalated blood is diluted with 3 parts of physiological saline, and smears are made on slides. The air-dried slides are treated with absolute ethanol for 2 min, dried, and placed in phosphate-citrate buffer of pH 3.2-3.6 for 1 min at 37°C. They are then rinsed in distilled water, and dried for storage or stained at once by either the iodination-coupled tetrazonium or the ferric ferricyanide reduction procedure. Adult hemoglobin is extracted by the buffer, so that red blood cells containing fetal hemoglobin give a much darker stain than those containing adult hemoglobin. The hemoglobin S of patients with sickle-cell anemia behaves like adult hemoglobin.     

Histochemische Beobachtungen am Saccus vasculosus der Forelle

Summary The saccus vasculosus of rainbow trout and brown trout, the latter caught in the wild, has been investigated by histochemical means. Isolated coronet cells and groups of them were found to be rather strongly but unspecifically stainable by alcian blue. A performic acid-aniline-aldehyde-thionine reaction demonstrated that such cells contain more disulfide groups than their nonstaining neighbours. This higher disulfide content and the stainability by alcian blue do not necessarily coincide with the presence of acid mucopolysaccharide, which was found in the cytoplasm of coronet cells in some cases. The hypothesis is discussed that cystine may be stored and used by coronet cells as a precursor of the acid mucopolysaccharide, which has been shown in the lumen of the organ.     

Effects of freezing and hardening on the sulfhydryl groups of protein fractions from cabbage leaves

Disc electrophoresis was used to separate water soluble proteins from hardy, non-hardy, and frost killed cabbage (Brassica oleracea var. capitata) leaves. Amidoschwarz staining failed to reveal any new bands as a result of hardening although the relative amounts of proteins in individual bands changed. Sulfhydryl groups in the protein bands were stained with 2,2-dihydroxy-6,6-dinaphthyl disulfide and labeled with ¹⁴C p-chloromercuribenzoate. Significant decreases in the sulfhydryl content of the total water soluble protein were found during hardening and as a result of frost death. The decrease during hardening was paralleled by a significant increase in the water soluble protein. There was a significant increase in the sulfhydryl content per unit high molecular weight protein but a decrease in the sulfhydryl content per total protein as a result of frost death. This was interpreted as evidence for intermolecular disulfide bond formation during freezing.     

A specific stain for sulfhydryl groups in proteins after separation by polyacrylamide gel electrophoresis

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.     

Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.     

The Barrnett-Seligman Sulfhydryl Reaction for Plant Meristems

Onion root tip meristems, fixed in 14 different fixatives representing ingredients and mixtures used in plant cytology, were tested with the Barrnett and Seligman histochemical procedure for protein-bound sulfhydryl groups. The relative intensity of staining was measured photometrically and the distribution of stain after each type of fixation described. Measurements indicated that conditions governing the staining of SH and S—S are not fully predictable; for example, fixation in saturated HgCl₂ enhances staining although inhibition was expected through mercaptide formation. Specificity of the reaction was further checked by treating fixed sections with known SH reagents. Partial blocking by such reagents as p-chloromercuribenzoate, and N-ethyl maleimide is apparently reversed by lengthy incubation in the 2,2'-dihydroxy-6,6-dinaphthyl-disulfide (DDD) reagent. Sulfhydryl oxidizing agents such as I-KI or chromic acid were either ineffective in blocking or could be reversed. For this reason and because previously reduced sections were proportionately better blocked than untreated ones it is suggested that the sulfhydryl reagent may open and then react with S—S bonds. Parallel runs indicate no difference in specificity between animal and plant tissues.     

Changes in accessibility of DNA to various fluorochromes during spermatogenesis

Accessibility of mouse testicular and vas deferens (vas) sperm cell DNA to acridine orange, propidium iodide, ellipticine, Hoechst 33342, mithramycin, chromomycin A3, 4'6-diamidino-2-phenylindole (DAPI), and 7-amino-actinomycin D (7-amino-AMD) was determined by flow cytometry. Permeabilized cells were either stained directly or after pretreatment with 0.06 N HCl. For histone-containing tetraploid, diploid, and round spermatid cells, HCl extraction of nuclear proteins caused an approximately sixfold increase of 7-amino-AMD stainability but had no significant effect on DAPI stainability. For these same cell types, the stainability with other intercalating (acridine orange, propidium iodide, ellipticine) and externally binding (Hoechst 33342, mithramycin, chromomycin A3) dyes was increased by 1.6- to 4.0-fold after HCl treatment. In sharp contrast, HCl treatment of vas sperm did not increase the staining level of 7-amino-AMD, DAPI, or propidium iodide but did increase the staining level for the other intercalating dyes (1.3- to 1.5-fold) and external dyes (1.3- to 1.9-fold). Elongated spermatids that contain a mixture of protein types including histones, transition proteins, and protamines demonstrated the greatest variability of staining with respect to type of stain and effect of acid extraction of proteins. In general, for nearly all dyes, the round spermatids had an increased level and tetraploid cells had a decreased level of stainability relative to the same unit DNA content of diploid cells. The observed differential staining is discussed in the context of chromatin alterations related to the unique events of meiosis and protein displacement and replacement during sperm differentiation.     

A histochemical investigation of the mechanism of Aldehyde Fuchsin staining of pancreatic B-cell granules

Summary The reaction mechanism by which Aldehyde Fuchsin selectively stains pancreatic B-cell granules is unknown. The participation of either insulin or proinsulin in the reaction is debatable; the stain may be bound by other components of the B-cell granule or its membrane. Sections of pancreas were stained with a variety of basic stains and specific histochemical reagents with and without appropriate blocking agents. No evidence for strong tissue anions associated with the B-cell granule could be found. Aldehyde Fuchsin staining was not abolished by lowering the pH below the point at which all known tissue anions should be protonated. There was no evidence that the Aldehyde Fuchsin staining solution itself generates reactive groups in the tissue. The results of this investigation support a non-ionic, possibly covalent mechanism for Aldehyde Fuchsin staining of pancreatic B-cell granules.