The use of 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B for the histochemical detection of lipid peroxidation in animal tissues — a microphotometric study

Summary The possibility of detecting lipid peroxidation histochemically by means of the 3-hydroxy-2-naphthoic acid/Fast Blue B (NAH-FBB) reaction was evaluated microspectrophotometrically. The procedure was modified in order to prevent exposure of tissue sections to lipid solvents. In fresh rat or mouse liver cryostat sections exposed in vitro to various prooxidant conditions (NADPH-Fe²⁺, NADPH-ADP/Fe³⁺, BrCCl₃-NADPH), a close correlation was found between the intensity of the NAH-FBB (blue-violet) stain and the amount of malondialdehyde — taken as biochemical index of lipid peroxidation — released in the incubation medium. Stain intensities obtained with NAH-FBB reaction were several fold higher than those obtainable with direct Schiff reaction — a previously used procedure — and better paralleled in time the appearance of lipid peroxidation in tissue. In particular, by means of selective delipidation it was observed that NAH-FBB reaction is remarkably more efficient than Schiff reaction in detecting protein and phospholipid-associated lipid peroxidation-derived carbonyl functions. The ability of the NAH-FBB reaction to reveal lipid peroxidation occurring in tissues in vivo was verified with animals intoxicated with prooxidant toxins, i.e. the haloalkanes bromotrichloromethane and carbon tetrachloride, and the glutathione-depleting agent bromobenzene. In livers from haloalkane-treated rats, NAH-FBB positivity provided with the specific absorption spectrum was observed in centrolobular regions. In bromobenzene-poisoned mice, NAH-FBB positivity with specific absorption was found — besides the liver — also in kidney (tubular epithelium) and lung (bronchiolar epithelium). The use of the NAH-FBB reaction is therefore suggested for the discrimination of cell types undergoing lipid peroxidation in vivo.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in prunus wood

Ice nuclei active at approximately −2°C and intrinsic to woody tissues of Prunus spp. were shown to have properties distinct from bacterial ice nuclei. Soaking 5-centimeter peach stem sections in water for 4 hours lowered the mean ice nucleation temperature to below −4°C, nearly 2°C lower than stems inoculated with ice nucleation-active Pseudomonas syringae strain B301D. Ice nucleation activity in peach was fully restored by air-drying woody stem sections for a few hours. The ice nuclei in woody tissue were inactivated between 40 and 50°C, but unaffected by treatment with bacterial ice nucleation inhibitors (i.e. NaOCl, tartaric acid, Triton XQS-20), sulfhydryl reagents (i.e. p-hydroxymercuribenzoate and iodine) and Pronase. Ice nuclei could not be dislodged from stems by sonication and were shown to be equally distributed in peach bud and internodal stem tissue on a per unit mass basis; outer and inner stem tissues were also indistinguishable in ice nucleation activity. Development of ice nuclei in immature peach and sweet cherry stems did not occur until midsummer and their formation was essentially complete by late August. Once formed the ice nuclei intrinsic to woody stems were stable and unaffected by seasonal changes in growth. The apparent physiological function of the ice nuclei is discussed in relation to supercooling and mechanisms of cold hardiness in Prunus spp.     

Histochemical observations on Pneumocystis carinii: Selective demonstration of honeycomb forms

Summary Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid — sodium bisulfite — resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Changes in abscisic acid and its β-D-glucopyranosyl ester levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The role of abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis was analysed. ABA and ABA ß-D-glucopyranosyl ester (ABA-GE) changes were determined in seeds and fruit tissues — placenta and mesocarp — during seed development, which was defined with eight embryo stages: from globular (stage 1) to mature embryo (stage 8). In whole seeds, ABA changes paralleled fresh and dry weight pattern curves and could be characterized by a high increase during embryo growth followed by a decrease as the seed matured and dehydrated. Moreover this dehydration phase led, at stage 8, to a new ABA distribution within the seed, preferentially into integument and embryo. Fruit tissue analyses provided new information about the ABA origin in seeds. ABA-GE levels were also measured and the results suggested different ABA metabolism in seed and fruit tissues.Abbreviations ABA abscisic acid - ABA-GE abscisic acid ß-D-glucopyranosyl ester - ABTS 2,2 — azino — bis (3 — ethylben-zthiazoline — 6 — sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA Ezyme linked immunosorbent assay - HPLC high performance liquid chromatography     

Light microscopical detection of antigens and lectin binding sites with gold-labelled reagents on semi-thin Lowicryl K4M sections: Usefulness of the photochemical silver reaction for signal amplification

Summary In the present study, we have investigated the applicability of semi-thin sections from low temperature Lowicryl K4M-embedded tissues for cytochemical labelling with protein A—gold and lectin—gold complexes. In order to ensure the best possible signal-to-noise ratio antibodies, protein A—gold and lectin—gold were applied in concentrations used for labelling at the electron microscope level. Furthermore, due to the lack of an appropriate chemical procedure for resin removal, untreated semi-thin sections were incubated. Under such conditions, semi-thin sections displayed either no visible staining or only a faint incomplete staining. However, following photochemical silver reaction, the latent or faint incomplete staining was rendered visible in most cases. It is concluded that the same block of Lowicryl K4M-embedded tissue and the same labelling reagents can be used for both light and electron microscopical cytochemical studies. At the light microscopical level, a high degree of structural and specific staining information is obtained. The reactivity of cellular components with antibodies or lectins is preserved even after years of storage of the blocks or slides containing semi-thin sections.     

Ultrastructural observations on the biogenic amines in the carotid and aortic-abdominal bodies of the human fetus

Summary The carotid and aortic-abdominal bodies of two human fetus at fifth month of pregnancy have been studied with the light and electron microscope. A personal variation of the glutaraldehyde ammoniacal silver (GA/S) method has been used, which consists in performing the silver reaction on the ultramicrotomical sections of the tissues, first fixed by glutaraldehyde perfusion and then included in Epikote 812.In the light microscope, the GA/S reaction is certainly positive in the aortic-abdominal bodies and it is negative or dubious in the carotid bodies. — In the electron microscope, however, the reaction is positive both in the aortic-abdominal and in the carotid bodies. — In the aortic-abdominal bodies, the silver precipitate accumulates into thick cytoplasmic granules, which have been shown to be NA-storing granules. — In the carotid bodies however, the silver precipitate accumulates into much smaller cytoplasmic granules, which are interpreted as 5-HT-storing granules.     

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane—isopropanol—water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.     

On the specificity of staining by Alcian blue in the study of human,foetal adenohypophysis

Summary The specificity of Alcian blue staining has been investigated on a material comprising the adenohypophysis from 26 human foetuses.Two distinct types of alcianophilic cells were observed. The first cell type appears about 28 mm CRL forming small groups at the epithelial-mesenchymal junction and beneath the anlage of the fibrous capsule. Besides its alcianophilic property the cell shows a pronounced maltase-resistant PAS-positivity. The alcianophilia is due to carboxylgroups — probably of proteins as no sialic acid could be detected. By the performic acid — Alcian blue method the cell seems to show a high content of SH and S-S groups.The second type of cell appears about 70 mm CRL and is mostly located singly. It shows a pronounced alcianophilia — probably due to sulphate groups and to some extent to carboxyl groups.The role of the alcianophilic properties of the cells in typing definite cells of the foetal adenohypophysis was discussed. — Finally the pitfalls caused by different fixatives and by the use of the general polychrome staining methods for the typing of pituitary parenchymal cells were discussed.This work was supported by grants from the Association for the Aid of Crippled Children, New York, and from the Danish State Research Foundation, Copenhagen.     

Effect of antler growth period on the chemical composition of velvet antler in sika deer (Cervus nippon)

The aim of this study was to provide basic information to allow improved scientific assessment of velvet antlers’ quality by investigating the change of chemical composition depending on antler growth period in sika deer (Cervus nippon). Twelve antlers harvested from sika deer stags (aged 4–5 years) by antler growth periods of 40 days (FDG) and 60 days (SDG) after the casting of antler buttons from the previous set were analysed to compare the chemical composition, such as crude protein, ash, ether extract, amino acid, collagen, glycosaminoglycans (GAGs), uronic acid and sialic acid. The weight of velvet antler in FDG was lower than that of the SDG (P<0.05). SDG had a higher (P<0.05) content of crude ash than FDG. FDG had a higher (P<0.05) content of crude protein than SDG. Amino acid composition was also higher in FDG than in SDG for all sections. The content of collagen was higher in SDG than in FDG for the middle and base (P<0.05) sections. However, collagen levels were exceptionally higher (P<0.05) in FDG than in SDG for the upper section. While the content of collagen was significantly higher (P<0.05) for the upper section than for the middle and base sections in FDG, this was significantly increased (P<0.05) downward from the upper to the base section in SDG. Uronic acid content was higher in FDG than SDG for all three sections but there was no significant difference between groups in the middle and base sections. The content of GAGs was significantly higher (P<0.05) in FDG than SDG for all three sections. The content of sialic acid was the same as that of GAGs. Consequently, in this study, velvet antler production was increased with the extension of antler growth period, but the contents of protein, total amino acid composition, GAGs, uronic acid and sialic acid were decreased and those of ash and collagen were increased. Therefore, it is expected that the quality of velvet antler may be decreased greatly by extension of antler growth period.     

Hyphal growth of Phytophtora cinnamomi on pine callus tissue

A procedure was developed which demonstrates the expression of differential resistance in pine callus tissues to the fungal pathogen Phytophthora cinnamomi Rands. Callus tissues were maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D and inoculated with hyphae of P. cinnamomi at 26°C in the dark. The number of intracellular hyphae was used as an index of resistance. Loblolly and loblolly × shortleaf pine hybrids were determined to be more resistant to infection and invasion by the fungus than were shortleaf and Virginia pine.Abbreviations (AL) loblolly pine—Alabama - (PL) South Carolina - (AS) shortleaf pine—Alabama - (CS) Georgia - (AV) Virginia pine—Alabama - (H1) loblolly × shortleaf pine hybrids—14–42 × 6-I-43 - (H2) I-523 × 6-D-8     

Pathway of indole metabolism by a denitrifying microbial community

The metabolism of indole in a mineral-salts medium inoculated with 9% anaerobically digested nitrate-reducing sewage sludge was studied. The sequential occurrence of four structurally-related compounds — oxindole, isatin, dioxindole, and anthranilic acid — was detected using high-performance liquid or thin-layer chromatography. Mass spectrometry and proton nuclear resonance were used to identify isatin and dioxindole isolated from the culture fluids. Prior exposure of the microorganisms to indole, oxindole, isatin, or anthranilic acid resulted in accelerated decomposition of these compounds in a pattern that was consistent with a proposed pathway for the metabolism of indole under denitrifying conditions.     

Morphochemical analysis of mucous cells in the skin and slime glands of hagfishes

Summary The mucous cells of the epidermis and slime glands in three species of Pacific hagfishes were studied histochemically to determine the presence or absence of acidic and neutral mucosubstances. Most mucous cells contained diastase-resistant PAS reactivity that varied in intensity. A few mucous cells contained diastase-labile substances exclusively.Acidic groups were detectable due to their stainability by several basic dyes which were utilized singly or in combination. Considerable species diversity of hagfish mucins was noted in their affinity toward azure A at pH 1.0, 2.0, and 3.0 depending on whether sections were viewed wet from the staining jar or viewed after dehydration and mounting. At pH 1.0, a few mucous cells were metachromatic under both conditions while the majority were unstained in both types of section. As the pH was raised, the majority of cells were metachromatic when viewed wet or mounted. Most mucous cells were reactive toward alcian blue at pH 1.0, aldehyde fuchsin and the high iron diamine reagents. Each of these reactions was absent following exposure of sections to acidic (0.1N HCl) methanol for 4 hours at 60°C while control sections exposed to unacidified methanol or acidified isopropanol for the same time and temperature resulted in no loss of staining.Sialidase-labile acidic groups were detected in the epidermal mucous cells of one species of hagfish. Following prior treatment of sections with Vibrio cholerae sialidase for 16–24 hours at 39° C, there was a reduction of metachromasia of the mucous cells produced by azure A at pH 3.0. Although confirmatory autoradiographic and biochemical data on hagfish mucosubstances are lacking, the histochemical staining results and their subsequent modifications by enzyme and chemical treatment indicate that the majority of these mucins contain sulfomucin while a few are composed of sialomucin. Similar results of previous histochemical and autoradiographic studies of epithelial secretions from higher animals correlated, in some instances, with the biochemical data for those mucins.Supported by Research Grant AM — 11064 of the United States Public Health Service.Recipent of a Lederle Medical Faculty Award, 1968–1971.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Detection of aldehydefuchsin-positive neurosecretory granules in the cells of the suprachiasmatic nucleus of the rat

Summary Application of dark field microscopy to sections fixed with picric acid — formalin and stained with crotonaldehydefuchsin allows the demonstration of neurosecretory granules in the neurones of the suprachiasmatic nuclei of normal rats.To whom reprint requests should be sentSupported by the Deutsche Forschungsgemeinschaft (Bo 392/4)     

The use of Quetol 651 for the post-embedding immunohistochemical demonstration of gamma-aminobutyric acid on semithin sections

Summary Quetol 651 was used as an embedding medium for the demonstration of aminobutyric acid (GABA) in semithin sections by the peroxidase—anti-peroxidase method. In order to demonstrate the immunoreactivity, the embedding medium was partially dissolved using absolute ethanol containing 0.8–1m NaOH or KOH for 5–7 min. The experimental procedure was elaborated by testing the GABAergic sites in the endings surrounding the small neurones of the anterior exterolateral nucleus of a mormyrid fish and in the pyramidal cells of the electrosensory lateral line lobe of gymnotoid fish by applying anti-GAD (glutamic acid decarboxylase) antiserum. To test the general validity of the use of Quetol 651, GABAergic sites were also identified in the central nervous system of an insect, the honey bee, with anti-GABA and anti-GAD antisera. The intensity of labelling revealed by immunoperoxidase applied to Quetol 651-embedded semithin sections, demonstrated high precision and gave good resolution for light microscopical observations.     

Ultracytochemical evidence for the presence of GERL in pinealocytes of the Mongolian gerbil (Meriones unguiculatus)

Summary Ultracytochemical reactions for the demonstration of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase, as well as zinc iodide-osmium tetroxide impregnation, revealed the existence of GERL (Golgi apparatus — Endoplasmic Reticulum — Lysosomes) in pinealocytes of the Mongolian gerbil (Meriones unguiculatus). The spatial arrangement of this structure was studied on thick sections using a goniometric stage. Although it was not possible to determine whether GERL in pinealocytes belongs to the Golgi apparatus or to endoplasmic reticulum, it can be concluded that its presence in studied cells signifies that they are considerably more active synthetically than has been believed to date.     

Approach to the analysis of diuretics and masking agents by high-performance liquid chromatography—mass spectrometry in doping control

The application of thermospray and plasmaspray high-performance liquid chromatography—mass spectrometry to the analysis of diuretics and probenecid has been investigated. The latter method gave better ionization efficiency than the former, and its response was optimized by altering the solvent composition: best results were obtained with water—methanol—acetonitrile—trifluoroacetic acid. Using different proportions of these solvents, three isocratic systems were developed to separate the compounds under study. The principal characteristic of plasmaspray positive-ion mass spectra was a protonated molecular ion and very little fragmentation was evident. In the negative ionization mode, the plasmaspray method gave mass spectra showing more fragmentation, which resulted in additional structural information. The ability of trifluoroacetic acid to form negative cluster ions precluded its use as a mobile phase component. The minimum detectable amounts determined by the analysis in the positive-ion mode was compound-dependent, but generally ca. 10–150 ng. In many cases the compounds could be detected in urine extracts.     

ULTRASTRUCTURAL CYTOCHEMISTRY : Enzyme and Acid Hydrolysis of Nucleic Acids and Protein

Selective extraction of specific cell components by enzyme or acid hydrolysis is possible from ultrathin sections for electron microscopy and parallel 2 µ sections for light microscopy of tissues fixed in formalin and embedded in a water-soluble polyepoxide, product X133/2097. Normal rat tissues fixed 15 minutes in formalin at 3°C are more rapidly digested by proteinases than those fixed for the same length of time at 20°C. Trypsin selectively attacks the nuclear chromatin and the ribonucleoprotein particles of the ergastroplasm, whereas mitochondria and zymogen granules resist tryptic digestion. Pepsin rapidly attacks the mitochondria and zymogen granules. The ergastoplasm and nucleus at first resist peptic digestion, but in time the entire cytoplasm and interchromatinic portion of the nucleus are attacked. Ribonuclease abolishes cytoplasmic basophilia in 2 µ sections, but parallel ultra-thin sections, stained with uranyl acetate and examined in the electron microscope, show no change in the ribonucleoprotein particles of the ergastoplasm. Desoxyribonuclease alone had no effect, but after pretreatment of the sections with pepsin or hydrochloric acid, desoxyribonuclease specifically attacked the nuclear chromatin. Nucleic acid-containing structures in the sections are gradually disintegrated by perchloric acid or hydrochloric acid.     

Hypergastrinaemia induced by acid blockade evokes enterochromaffin-like (ECL) cell hyperplasia in chicken,hamster and guinea-pig stomach

Summary Treatment of chickens, hamsters and guinea-pigs with large doses of the long-acting antisecretory agent omeprazole for 10 weeks resulted in elevated serum gastrin levels and in increased stomach weight and mass of oxyntic mucosa. Also the antral gastrin cell density was increased. Another striking effect was the hyperplasia of the histamine-producing enterochromaffin-like (ECL) cells — a prominent endocrine cell population with unknown function — in the oxyntic mucosa. Accordingly, the gastric mucosal histamine concentration and rate of histamine formation were increased in all three species. The results suggest that marked and long-lasting suppression of acid secretion leads to elevated serum gastrin levels and diffuse ECL cell hyperplasia not only in the rat, as previously seen, but also in the chicken, hamster and guinea-pig; this hyperplasia is associated with accelerated histamine formation in all three species. The following sequence of events is suggested to occur in mammalian as well as submammalian vertebrates: suppression of acid secretion — hypergastrinaemia — ECL cell hyperplasia.