Peroxidase and pseudoperoxidase reactions in relation to sudanophilia

Summary A selective staining of hemoglobin in erythroid cell series was achieved by use of Sudan Black B (modified method of Sheehan and Storey) if optimal amount of hydrogen peroxide was added to the staining mixture. The effect of some inhibitory agents (KCN, wet heat, pH) on this staining as well as on the Lepehne's pseudoperoxidase reaction for hemoglobin was similar. Both reactions were more resistant to these factors than the peroxidase reactions and sudanophilia in granulocytes in which both could be blocked by the pretreatment with absolute methanol. Moreover the effect of some extraction procedures for lipids on both myeloperoxidase reactions and sudanophilia was investigated. The results support the view that the sudanophilia in granulocytes is due to their peroxidase activity and for the staining of hemoglobin by use of Sudan Black B with H₂O₂ its pseudoperoxidase activity is responsible. In addition the effect of the substitution of phenolphosphate by dihydroxybenzenes on granulocyte sudanophilia is reported.     

长薄鳅外周血细胞的显微结构和细胞化学特征研究

长薄鳅外周血细胞可分为红细胞、中性粒细胞、单核细胞、淋巴细胞和血栓细胞.在数量上,中性粒细胞、单核细胞、淋巴细胞和血栓细胞占白细胞总数的百分比分别是17.06%、5.83%、28.16%和48.94%.细胞化学染色显示所有白细胞均含有糖原物质,所有红细胞均不含酸性磷酸酶,中性粒细胞、单核细胞、淋巴细胞和血栓细胞均含有酸性磷酸酶.非特异件酯酶染色显示单核细胞呈阳性反应,中性粒细胞、淋巴细胞和血栓细胞均为部分呈阳性反应.所有细胞的碱性磷酸酶、过氧化物酶、苏丹黑显色反应均呈阴性.     

Changes in lipid content during the post-hatching development of the brain of altricial birds

Summary The post-hatching development of the brain was studied in three altricial birds (those whose development is completed after hatching), namely, the domestic pigeon, house swift and house sparrow, to assess the state of maturity of the brain at the time that the fledglings leave the nest. Malurity is related to the process of myelination and, therefore, Sudan Black was chosen as it is a sensitive indicator of myelin lipids. On hatching, it was found that sudanophilia was very low in all three birds, indicating a lack of maturation. However, between 15 and 25 days in the pigeon and swift and between 8 and 14 days in the sparrow, there was a steady increase in Sudan Black staining. By the time the birds left their nests, the staining was similar to that of adults, thus indicating that the process of myelination was complete and that the brains were nature.     

Sudanophilia and lipids in the blood and marrow cells

Summary The method of Sheehan and Storey, used for demonstrating stable Sudanophilia of neutrophils in smears, was modified by substituting other alcohols (from methanol to amyl alcohol) for ethanol, and the effect of this on the resulting staining reactions was investigated. The intensity of the staining of the neutrophil granules decreases with increasing number of carbon atoms in the alcohol, with a gradation of staining from black to yellow, while, at the same time the staining of the erythrocytes increases. The best solvent for Sudan black B for haematological purposes is methanol. This solvent was further used for investigations on various procedures for detecting so-called masked sudanophilia. The most constant and interesting results were obtained after the action of mercuric ions on smears fixed in formalin vapour. Under certain conditions, a characteristic binding of different fractions of Sudan black B on different blood cells can be attained, so that neutrophil granules are stained brown, monocytes mostly grey, lymphocytes, platelets, megakaryocytes, plasmocytes and perinuclear areas of normoblasts blue. The present experiments with various substrates indicate that demasked blue staining in the cytoplasm may be conditioned by phospholipids, although a reaction with other components of the cytoplasm cannot be excluded. The method described may be useful in haematology for studying cytogenetic relations in the monocytic and granulocytic series.Technical Assistance: V. LodrovÁ.     

Partial deficiency of eosinophil peroxidase

A new case of partial eosinophil peroxidase deficiency is reported. It was identified and elaborated using an automated flow-cytochemical analyzer. The findings were confirmed by conventional cytochemistry on blood smears: decreased staining intensity in peroxidase and Sudan Black staining. These results are in agreement with other reports on this extremely rare peroxidase deficiency, which probably is of no clinical relevance.     

Sensitive assay systems for detection of hemoglobin with 2,7-diaminofluorene: histochemistry and colorimetry for erythrodifferentiation

Sensitive and rapid assays, colorimetry and histochemistry, for hemoglobin in erythroid cells are established. The assays are based on pseudoperoxidase activity of hemoglobin using 2,7-diaminofluorene as a hydrogen donor for the peroxidase, instead of benzidine which is widely benzidine which is widely used for the detection of small amounts of hemoglobin but which is a potent carcinogen and has been banned from laboratory use. In the presence of hydrogen peroxide, hemoglobin catalyzes the formation of a blue compound (fluorene blue), which has a broad absorption band between 500 and 690 nm with a peak at 610 nm, from 2,7-diaminofluorene. The reagent is safe to use in the laboratory. The methods could be applied to the detection of hemoglobin in Friend erythroleukemia cells induced to cell differentiation along the erythroid pathway by dimethyl sulfoxide.     

Some aspects of the value of Sudan Black B in lipid histochemistry.

The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films. Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (2:1) before staining resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lamber-Beer, Proteins were however, also coloured by the dye. The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show a specific binding. The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

Thin layer chrornatography and histochemistry of Sudan Black B

Summary Thin layer chromatography of commercial Sudan Black B on silica gel with chloroform-benzene (11) as the developing solvent reveals two blue main fractions with R_f values of 0.49 and 0.19, SBB-I and SBB-II respectively. Furthermore at least eighteen secondary fractions or impurities have been found. SBB-I and SBB-II were isolated and purified by preparative thin layer chromatography. Commercial Sudan Black B consists of about 20 p.c. SBB-I, 60 p.c. SBB-II and 20 p.c. secondary fractions.From spectrophotometrical and histochemical investigations it appeared that SBB-I stains lipids more pronounced than SBB-II; moreover SBB-I is more specific for neutral lipids than SBB-II, which fraction may also stain some proteins and acid mucopolysaccharides. Contrary to SBB-II the staining with SBB-I is fairly independent of pH. Finally, the colour of SBB-II changes under the influence of light and air, while SBB-I is much more stable.A physico-chemical study of the nature of SBB-I and SBB-II, including spectrophotometry, chromatography, infrared spectroscopy and chemical analysis revealed, that SBB-II is a basic dye, while SBB-I in spite of the structural resemblance behaves as a neutral one, dissolving therefore better in neutral lipids.As yet the chemical composition of SBB-I and SBB-II, and the relation to the scheme of synthesis of Sudan Black B has not been solved. The unspecificity of lipid staining by Sudan Black B is due to the basicity of SBB-II, and to the instability of this dye toward light and air. Moreover the some eighteen impurities may have some influence on the staining properties. The question of solubility or adsorption processes in the case of lipid staining by Sudan dyes is at least partially answered by the proposition of a dissolving fraction SBB-I and an adsorbed fraction SBB-II. The changing absorption spectra by the corresponding solvatochromic and metachromatic effects may give information about the nature of the lipids stained.     

Cytochemical studies of normal feline blood and bone marrow cells

Blood and bone marrow cells of ten clinically healthy cats were stained for alkaline phosphatase (ALP), peroxidase (PO), chloroacetate esterase (CAE), alpha-naphthyl butyrate esterase (NBE), sudanophilia, and periodic acid-Schiff (PAS) reaction. Mature neutrophils in blood and bone marrow were devoid of ALP and NBE, but exhibited modest to strong PO, CAE, sudanophilia, and PAS reaction. In bone marrow, sudanophilia, PO, and CAE were prominent at the promyelocyte stage and diminished with cellular differentiation and maturation, while PAS reactivity increased with cell maturation usually from the myelocyte stage onwards. Myeloblasts were negative for all cytochemical reactions, but some large unidentifiable cells reacted strongly for ALP. Eosinophils were slightly reactive for ALP, CAE, and PAS, but not for PO, sudanophilia, and NBE. Basophil granules stained strongly for CAE, revealed PAS positivity, and stained negatively for PO, NBE, ALP, and sudanophilia. Slight ALP activity was detected in the intergranular cytoplasm of basophils. Lymphocytes and monocytes, with few exceptions, stained negatively. An occasional lymphocyte revealed slight globular NBE activity (NaF-resistant) and diffuse PAS reaction, while an occasional monocyte contained a few PO-positive and sudanophilic granules. Monocytes reacted modestly, whereas bone marrow macrophages reacted strongly for NBE (NaF-sensitive). Cells of the erythroid series stained negatively for all cytochemical reactions, megakaryocytes were PAS-positive, and platelets gave positive reactions for PAS and CAE.     

Oil-soluble dyes for marking Spodoptera frugiperda (Lepidoptera: Noctuidae)

Although various biological aspects of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) have been examined, adult movement and dispersal of this insect pest is not well understood. Release-recapture techniques by using marked insects is a useful approach for dispersal studies; however, the marking technique should not significantly affect insect biology or behavior. Therefore, the effect of different concentrations of oil-soluble dyes (Solvent Blue 35 [C.I. 61554], Sudan Red 7B [C.I. 26050], Sudan Black B [26150], Sudan Orange G [C.I. 11920], and Sudan I 103624 [C.I. 12055]) on development, mortality, and fecundity of S. frugiperda was evaluated. Dyes were added to artificial diet used to feed larvae. Larval and pupal development and mortality, adult longevity, and female fecundity were evaluated. High concentrations (400 and 600 ppm) of all dyes led to longer larval and pupal stages. Adult life span and number of eggs were not affected by the dyes. Sudan Red 7B marked both adults and eggs very well. Solvent Blue 35 marked both adults and eggs, but the blue-marked eggs could not be distinguished from some bluish eggs laid by nonlabeled females. Adults and eggs were not adequately marked by the Sudan Black B, Sudan Orange G, and Sudan I 103624 (a yellow dye).     

Cytochemical aspects of Mercenaria mercenaria hemocytes.

The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.     

Some aspects of the value of Sudan Black B in lipid histochemistry

Summary The lipid dye Sudan Black B, as generally used to demonstrate lipids in the interior of the cell nucleus, was studied with regard to its staining properties for isolated nuclei in relation with its chromatographic characteristics in solution, as well as with a model system consisting of lipid containing polyacrylamide films.Isolated nuclei are stained with Sudan Black B dissolved in ethylalcohol, only when the dye-solution is at least one month old. Extraction with chloroform-methanol (21) before stainig resulted in a decrease of 35% in intensity. Treatment with proteolytic enzymes and DNA-se caused a complete disappearance of the staining capacity. The binding of Sudan Black B with phospholipids enclosed in the form of liposomes in modelfilms when stained with this dye in ethylene glycol obeys the law of Lambert-Beer. Proteins were however, also coloured by the dye.The chromatographic experiments showed that the dye is built up from two main and a number of secondary products. The secondary products which increase by aging of the dye-solution, change the spectrophotometric properties of the total dye and show aspecific binding.The conclusion was reached that on the basis of a positive reaction with Sudan Black B no definite conclusions can be drawn about the presence of lipids in the interior of the cell nucleus.     

A comparison of methods using diaminobenzidine (DAB) to localize peroxidases in erythrocytes, neutrophils, and peroxidase-antiperoxidase complex.

Reactions using diaminobenzidine (DAB) to localize the enzyme peroxidase in neutrophils and peroxidase-antiperoxidase (PAP) complex during immunological staining are usually performed in Tris-HCl or phosphate buffer at pH 7.2-7.6. However, DAB solutions at pH 7.2-7.6 often demonstrate erythrocyte pseudoperoxidase as well. By lowering the pH of the DAB solutions, it is possible to selectively suppress the reactivity of pseudoperoxidase while maintaining optimal reactions in neutrophils and PAP complex. For this purpose we recommend ammonium acetate-citric acid buffer at pH 5.5 (pH 5.0-6.0) containing 44 mg DAB per 100 ml buffer and 0.003%-0.03% with respect to H2O2.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

A Comparison of Methods Using Diaminobenzidine (Dab) to Localize Peroxidases in Erythrocytes, Neutrophils, and Peroxidase-Antiperoxidase Complex

Reactions using diaminobenzidine (DAB) to localize the enzyme peroxidase in neutrophils and peroxidase-antiperoxidase (PAP) complex during immunological staining are usually performed in Tris-HCI or phosphate buffer at pH 7.2-7.6. However, DAB solutions at pH 7.2-7.6 often demonstrate erythrocyte pseudoperoxidase as well. By lowering the pH of the DAB solutions, it is possible to selectively suppress the reactivity of pseudoperoxidase while maintaining optimal reactions in neutrophils and PAP complex. For this purpose we recommend ammonium acetate—citric acid buffer at pH 5.5 (pH 5.0-6.0) containing 44 mg DAB per 100 ml buffer and 0.003V-0.03% with respect to H₂O₂.     

Characteristics on staining of leukocytes in experimental animals]

The staining characteristics of the peripheral blood cells from mouse, rat, guinea pig, rabbit, dog, marmoset and monkey were studied. In marmoset, it is easy to distinguish neutrophils from eosinophils by using the phosphate-buffered solution of pH 5 or 6. It was found in the special staining methods that neutrophil granules showed intense peroxidase and Sudan black B reactions in marmoset in comparison with those in the other species of experimental animals. Neutrophil granules rabbit was, however, intensely stained with esterase and acid phosphatase.     

Taenia taeniaeformis: early inflammatory response around developing metacestodes in the liver of resistant and susceptible mice II. Histochemistry and cytochemistry

Female BALB/cJ (resistant), C3H/HeJ (intermediate resistant), and C3H/HeDub (susceptible) inbred mice, 4-5 wk old, were infected with Taenia taeniaeformis. Liver sections were stained for the enzymes acid phosphatase, beta-glucuronidase, and peroxidase. Eosinophils present around the parasite were identified by the ethanolic Congo red method. Possible gross changes in lipid metabolism in the hepatocytes surrounding the parasite were investigated with the Sudan black B method. The results of observations made by light microscopy were: (1) beta-glucuronidase activity above background levels was observed only in the hepatocytes around the parasite in BALB/cJ mice at 4, 5, and 6 days postinfection (PI); no reaction was observed in the other 2 strains of mice studied; (2) acid phosphatase activity was very strong at 2, 3, and 4 in the 3 strains of mice while this reactivity was weak at 5 and 6 days PI; (3) the cytoplasm of the hepatocytes around the metacestode stained more heavily with Sudan black B than other hepatocytes; and (4) the presence of eosinophils appearing at 3 days PI around the parasite in all 3 strains of mice was demonstrated by staining with Sudan black B, the substrate of peroxidase, and Congo red. Infected C3H/HeJ and BALB/cJ mice had higher numbers of liver eosinophils than infected C3H/HeDub mice throughout the observation time. The present results suggest 2 conclusions: (1) a parasite-liver interaction occurs as is evident by hepatocyte changes in beta-glucuronidase activity and Sudan black B staining, and (2) resistance to the early stages of T. taeniaeformis is associated with the appearance of eosinophils.     

Iron content correlates with peroxidase activity in cysteamine-induced astroglial organelles.

A subpopulation of astrocytes in periventricular brain regions and in cysteamine-treated neuroglial cultures contains cytoplasmic granules that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. The autofluorescence and pseudoperoxidase activity are consistent with the presence of porphyrins and heme iron, respectively. In the present study, we employed diaminobenzidine cytochemistry, transmission electron microscopy, and energy-dispersive X-ray microanalysis (electron microprobe) in an attempt to correlate fine structure with the peroxidase activity and elemental composition of the cysteamine-induced inclusions in cultured astrocytes. In osmicated preparations, these membrane-bound inclusions varied greatly in size, were round or ovoid in shape, and exhibited an intensely electron-dense granular matrix. In non-osmicated preparations, many inclusions exhibited internal membranous partitions producing complex subcompartmentalization. Diaminobenzidine reaction product, indicative of endogenous peroxidase activity, was occasionally observed distributed diffusely throughout the granule matrix. More commonly, peroxidase activity was restricted to specific intraorganellar compartments. Elemental iron was detected in the inclusions by electron microprobe analysis. The presence and concentration of iron in these organelles correlated closely with the presence and intensity of diaminobenzidine staining, suggesting that redox-active iron mediates the pseudoperoxidase reactions in these cells. Cysteamine-induced derangements of porphyrin-heme biosynthesis may be responsible for the proliferation of iron-containing gliosomes in these astrocytes.     

American paddlefish leukocytes demonstrate mammalian‐like cytochemical staining characteristics in lymphoid tissues

American paddlefish Polyodon spathula leukocytes demonstrated cytoplasmic staining patterns very similar to mammalian leukocytes when stained with acid phosphatase, α‐naphthyl butyrate esterase and β‐glucuronidase. American paddlefish monocytes, lymphocytes and granulocytes stained positive for acid phosphatase. Monocytes stained positive for α‐naphthyl butyrate esterase. Lymphocytes that stained positive for α‐naphthyl butyrate esterase were designated type A. Lymphocytes that stained positive with antibodies to the L chain of white sturgeon Acipenser transmontanus immunoglobulin (Ig) were designated type B. Type A and type B lymphocytes stained positive for β‐glucuronidase. All leukocytes observed were negative for Sudan Black B. Monocytes, lymphocytes and granulocytes were present in the renal haematopoietic tissue, spleen, thymus, pericardial myeloid tissue, lamina propria of the spiral valve, and in meningeal myeloid tissue located dorsal to the brain, at the base of the brain and around the notochord. Peyer's patches were present in the gut. Morphological characteristics of leukocytes stained with Wright's and haematoxylin and eosin and appeared very similar to those of other fish species.     

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.