Silver Methenamine Method Research Articles

Histological, immunohistochemical and ultrastructural features of polyglucosan bodies in uterine smooth muscle of pet rabbits (Oryctolaguscuniculus)

We document the frequency and morphological and immunohistochemical features of inclusion bodies in uterine smooth muscle cells in 56 (76%) of 74 investigated pet rabbits (Oryctolagus cuniculus). Inclusion bodies began to appear at the age of 2 years and their frequency increased with age (P = 0.047, r = 0.33). They ranged from 5 to 20 μm in diameter, were slightly basophilic to amphophilic with well-delimited oval bodies in haematoxylin and eosin-stained tissue sections and formed in the cytoplasm of the uterine smooth muscle cells with displacement of the cell nuclei. The inclusion bodies were positive with periodic acid–Schiff, Best's carmine, Lugol's iodine and Grocott's methenamine silver methods. They were immunoreactive to a monoclonal antibody raised against human polyglucosan and negative with monoclonal antibodies for several intermediate filament proteins. Electron microscopy revealed that they were non-membranous structures composed of electron-dense amorphous material. The morphological, histochemical, immunohistochemical and ultrastructural features of the inclusion bodies in the rabbi uteri were similar to those of human polyglucosan bodies (PGBs). PGBs appear to occur at a high frequency in the uterus of rabbits, which are known to be susceptible to uterine diseases.

Application of platelet-rich plasma and platelet lysate in the treatment of experimental lymphocutaneous sporotrichosi.

Background and Purpose:Sporotrichosis is a subcutaneous and chronic fungal infection that is caused by a dimorphic fungus, namely Sporothrix schenckii sensu lato. Lymphocutaneous sporotrichosis is the most clinical form, which accounts for nearly 80% of the cases of cutaneous sporotrichosis. Platelets contain several substances with antimicrobial properties. Regarding this, the present study was performed to investigate the effect of blood-based biomaterials, especially platelets in the treatment of lymphocutaneous sporotrichosis.Materials and Methods:This study was performed on 12 golden hamsters, divided into three groups of control, platelet-rich plasma, and platelet lysate. For the purpose of the study, Sporothrix conidia suspension was injected subcutaneously on the back of the animals. After the induction of subcutaneous lesions, the Gomori methenamine silver method was applied to verify lymphocutaneous sporotrichosis. Subsequently, plasma-rich platelet and platelet lysate were injected into the created lesions in the animals in 3-day intervals (due to the short lifetime of platelets). In the final sage, skin tissue samples were examined to check for the presence of yeast cells and their quantification.Results:The data were indicative of the presence of yeast cells with/without bud in the tissue of lymphocutaneous sporotrichosis lesions in the infected animals. Histological investigation revealed that each of the two biomaterials under study (i.e., plasma-rich platelet and platelet lysate) played a positive role in the removal of the yeast cells of sporotrichosis.Conclusion:The results of this study showed that both plasma-rich platelet and platelet lysate were able to effectively prevent from the progression of cutaneous sporotrichosis. Accordingly, much attention has been given to new therapies, including treatment with blood-derived biomaterials.

Polyglucosan Bodies in the Prostatic Stromal Smooth Muscles of Aged Dogs.

Polyglucosan bodies (PGB) in the prostate of aged dogs without neurological signs were examined by light microscopy, histochemistry and immunohistochemistry. Prostatic PGB were round or oval and slightly basophilic. Most of the bodies were situated within the stromal smooth muscle cells. PGB were intensely positive for PAS, Best's carmine, Lugol's iodine and Grocott's methenamine silver method. Moreover, canine prostatic PGB were immunoreactive for monoclonal antibodies raised against human polyglucosan. The frequency of PGB in the smooth muscle cells was significantly correlated with the age of dogs. The occurrence of PGB in the canine prostate might be a non-specific finding related to ageing.

A study of interobserver reproducibility of morphologic lesions of focal segmental glomerulosclerosis

The morphology of focal segmental glomerulosclerosis (FSGS) includes collapsing, cellular, and sclerosing forms. The Columbia Working Classification of FSGS divides these into collapsing (COLL), cellular (CELL), tip lesion (TIP), perihilar (PH), and not otherwise specified (NOS) morphologic forms. This study examined the ability of renal pathologists to classify FSGS using single light microscopic images of glomeruli as a uniform data set. Sixty-one digital images of individual glomeruli with FSGS, stained by periodic acid-Schiff or Jones methenamine silver methods, were classified independently by six specialist renal pathologists. Diagnostic consistency was quantified using the kappa statistic for nominal categories. Agreement for 366 diagnoses by six observers was 75.2 % with a kappa value of 0.676. Six of six observers agreed in 31 of 61 cases (50.8 %) and four or more in 53 cases (86.9 %). Respective kappa values ranged from moderate to good: COLL 0.77, CELL 0.53, TIP 0.76, PH 0.84, and NOS 0.60. Capillary retraction with lobular expansion, hypercellularity, and sclerosis in the same glomerular segments, and the location of segmental lesions were sources of diagnostic inconsistency. The morphologic forms of FSGS defined by the Columbia system are reproducible between observers and have a low probability of confusion between forms. Individual glomeruli may have overlapping features of more than one form of FSGS.

Improved Selectivity and Sensitivity in the Visualization of Neurofibrillary Tangles, Plaques and Neuropil Threads

Stain sensitivity is a key factor in estimating the frequency of plaques and neurofibrillary tangles in Alzheimer's disease (AD), making it essential that the sensitivity and selectivity of detection methods for identifying these lesions is maximized. Several new, improved techniques have recently been described, although these methods have not been compared quantitatively with those techniques currently recommended for use in standardized diagnostic protocols. In the present study, eight different stains were examined for their selectivity and sensitivity in detecting plaques and tangles in serial tissue sections from AD and control brains. Techniques compared were immunohistochemistry for tau and β-amyloid, thioflavin S, nickel peroxidase method, and four silver impregnation techniques (Gallyas silver iodide, Campbell-Switzer-Martin, Garvey's modified Bielschowsky and methenamine silver methods). Among these eight staining techniques, the nickel peroxidase proved the most reliable method for the demonstration of the histopathological lesions of AD. This method labels all morphological types of plaques and tangles within a single tissue section, and provides several advantages for the analysis of lesion progression and distribution.

Age-related deposition of glia-associated fibrillar material in brains of c57BL/6 mice

With advancing age, clusters of unusual granules appear in the brains of C57BL/6 (B6) mice. At the light, confocal laser and electron microscopic levels, the granules represent aggregations of fibrillar material often associated with astrocytes. The fibrillar material is largely free of normal organelles and has been located within astrocytic somata and processes, although in many cases the material is found in the neuropil and is surrounded by a discontinuous membrane. The deposits occur predominantly in hippocampus, but also in piriform cortex, cerebellum and less frequently in some other brain regions. They become evident about six months of age and increase markedly in both number and size thereafter. Incidence of the deposits varies greatly among inbred mouse strains. At six to 12 months of age, granules are abundant in male and female B6, and are absent in BALB/c, CBA, DBA/2 and A mice. In hybrid strains with a B6 background the deposits are also present and thus appear to manifest dominant genetic heritability. Similar granular structures have been described in adult brains of the senescence accelerated mouse and have been noted, albeit very rarely, in aged mice from other strains. While immunostaining of the granules with several polyclonal antisera was found by preabsorption with antigens to be non-specific, immunolabeling with monoclonal antibodies to heparan sulfate proteoglycan core protein and to laminin suggest these or related molecules as components of the fibrillar material. The presence of glycosaminoglycans is supported by staining with periodic acid-Schiff and Gomori's methenamine silver methods. The functional significance of the murine deposits is not yet clear. The deposits do not represent senile plaques with β-amyloid deposition, but they might mimic the deposition of extracellular matrix molecules that is hypothesized to be a precursor condition for plaque formation and cerebral amyloidosis. Furthermore, the genetic differences in the incidence of the fibrillar deposits has potential to model aspects of familial neurodegenerative diseases.

Glycoconjugates in the cochlea as revealed by the silver methenamine method

The glycoconjugates in the cochlea of the guinea pig were studied by staining samples by the silver methenamine method as well as after periodic acid-Schiff (PAS) staining. Results obtained by the two methods were similar but not identical. The silver methenamine method was much better in terms of resolution. However, this method of staining seemed less specific than the PAS reaction. When the silver methenamine method was used, the tectorial membrane and outer hair cells were specifically stained. Two types of fibrils were observed in the tectorial membrane. Thick fibrils were located in the fibrous layer. Thin fibrils were situated in the marginal band, the cover net, Hensen's stripe and the fibrous layer. The thick and thin fibrils appeared to correspond to type A and type B protofibrils, respectively. The outer hair cells were found to contain strongly stained particles which, presumably, consisted of glycogen. The basement membrane of the capillaries in the stria vascularis also gave a positive reaction, while that of other capillaries was essentially unstained. This finding suggests structural differences between these capillaries.

Glycoconjugates in the vestibular organs as revealed by the silver methenamine method

The glycoconjugates in the vestibular organs of the guinea pig were studied after staining by the silver methenamine method and by the periodic acid-Schiff (PAS) reaction. The organic matrix of otoconia, otolithic membranes and cupulae were stained to the same degree by the PAS reaction. In contrast, the mineralizing and non-mineralizing matrices were clearly distinguished by the silver methenamine method. The otoconia were surrounded by an intensely stained organic matrix, while the otolithic membranes and cupulae were moderately stained. This histochemical difference suggests that the positively stained organic matrix of otoconia is not identical to the otolithic membranes and cupulae in terms of its biochemical composition. The strongly stained material may play an important role in turnover of calcium in otoconia. The contact areas between type I hair cell and nerve calyx were contained silver methenamine-positive material which is probably involved in adhesion of these cell membranes.

The fine structure of the guinea pig tympanic membrane with special reference to the fiber arrangement.

The tympanic membrane of the guinea pig was studied by electron microscopy. The pars tensa was composed of three layers, i.e., the outer epidermis, the middle lamina propria and the inner mucosal membrane. The pars flaccida was negligible in size. The fibers in the lamina propria of the tympanic membrane were not clearly observable by conventional electron microscopy. With the silver methenamine method the fibers were more intensely stained than the epithelial cells and could thus be distinctly demonstrated by this method. In the inferior larger portion of the pars tensa, the lamina propria contained the outer radial and the inner circular fiber bundles. The radial fiber bundles were thicker and more densely packed than the circular ones. This fact indicates that the radial fibers form the major skeleton of this portion. The superior smaller portion of the pars tensa contained several layers of irregularly arranged fiber bundles in the lamina propria and was considerably thicker than the inferior larger portion.

Comparison between histochemical and immunohistochemical methods for diagnosis of sporotrichosis.

To compare the efficacy of histochemical and immunohistochemical methods in detecting forms of Sporothrix schenckii in tissue. Thirty five cutaneous biopsy specimens from 27 patients with sporotrichosis were stained by histochemical haematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver methods and an immunohistochemical (avidin-biotin complex immunoperoxidase) (ABC) technique associated with a newly produced rabbit polyclonal antibody anti-Sporothrix schenckii. A total of 29 (83%) cases were positive by the ABC method used in association with anti-Sporothrix schenckii rabbit polyclonal antibodies. Histochemical methods, using silver staining, periodic acid Schiff, and conventional haematoxylin and eosin detected 37%, 23%, and 23% of forms of S schenckii, respectively. The ABC technique was significantly more reliable than periodic acid Schiff and silver staining techniques. It is concluded that immunostaining is an easy and rapid method which can efficiently increase the accuracy of the diagnosis of sporotrichosis in human tissue.

Electron microscopical demonstration of thiols and disulphides in the porcine epidermis

This study describes the electron microscopical distribution of free thiols and disulphides in the epidermis of the domestic pig and the wild boar, as compared to light microscopical histochemistry. With the silver methenamine method, silver labelling of thiols was clearly achieved on the keratohyalin and cytofilament accumulations in the cells of the living epidermis and the plasma membrane of granular cells. To a certain extent, the envelope and cytoplasm of young corneocytes reacted equally intensively. Disulphides were very abundant in the filaments, keratohyalin granules, and cell envelope of granular cells, and, particularly, in the envelope (marginal band) of corneal cells; the latter structure being distinctly delineated from the background. As a specific feature, the viable epidermis of the wild boar stained strongly for disulphides. The results obtained are discussed in view of actual concepts of epidermal keratinization and corneal cell function.

A silver methenamine method for the demonstration of biologic calcification sites

Much work has been carried out in our laboratories with the periodic acid-thiocarbohydrazide silver methenamine(PATS) reaction, an improved variation of the periodic acid-Schiff(PAS) or periodic acid-thiocarbohydrazide(TCH)-osmium tetroxide(PATCO) reactions. The PATS stain was used in many of our studies on the vascularization of hydroxylapatite/plaster of Paris implants for bone restoration. It was very useful in demonstrating the Type III collagen or reticulin of the pericapillary reticular network of the newly formed blood vessels. In the course of these studies the improved ability of silver methenamine solutions applied under microwave irradiation to deposit silver at sites of calcification or certain calcium compounds in our specimens was noted. Much more silver was deposited at these sites in femur sections of 5 day old mice after application of 1% silver methenamine soln (pH 9.6) for 1 min at 2450 MHZ and reducing with TCH for 30 seconds (Figs. 1,3,4) than by application of 5% silver nitrate for 30 minutes under bright light and reduction for 2 1/2 minutes with 5% sodium thiosulfate (Fig. 2).

The prevalence of amyloid (A4) protein deposits within the cerebral and cerebellar cortex in Down's syndrome and Alzheimer's disease

The extent of amyloid deposition within the cerebellum and the cerebral cortex was assessed and compared, using anti-amyloid protein (A4) immunostaining and a novel methenamine silver method, in 20 patients aged between 60 and 77 years with Alzheimer's disease (AD), 29 patients aged between 13 and 71 years with Down's syndrome (DS), 26 demented patients with disorders other than AD and DS and in 20 non-demented elderly individuals of age range 60-102 years. In AD, amyloid deposits were noted in the cerebellar cortex in 90% of patients and in the meningeal vessels of the cerebellum in 80% of patients. In DS, amyloid deposits were seen in the cerebellar cortex in 82% of patients over 30 years of age and was universal in patients over 50 years of age. Overall, in DS, amyloid deposits were present in the meningeal vessels of the cerebellum in 79% of patients, but were present in 94% of those patients over 50 years of age. The sites of amyloid deposition in the cerebellar cortex were (poorly) detected by lectin histochemistry (Concanavalin A binding) in only 40% of patients with AD and 43% of all patients with DS (69% of those over 50 years of age). No amyloid deposits were seen in either the cerebellar cortex or its meningeal vessels in any of the 20 non-demented elderly individuals nor in any of the non-Alzheimer demented patients. The cerebellar amyloid deposits were never associated with a neuritic change [i.e. as characterised by the presence of (tau-positive) paired helical filaments (PHF)] and neurofibrillary tangles were seen only in a few cells of the dentate nucleus in a single patient with AD and in three of the elderly DS patients. Amyloid deposits were numerous in the cerebral cortex of all patients with AD and in all, except the 13-year-old patient, with DS. In all the AD patients and in most of the DS patients over 30 years of age, many of the cerebral cortical amyloid deposits were associated with neurites and were strongly recognised by lectin histochemistry. Amyloid deposits were present within the meningeal vessels of the cerebral cortex in 75% patients with AD and 72% of patients, over 30 years of age, with DS (82% of those over 50 years of age). These data indicate that the process of amyloidosis in AD and in elderly DS patients is not restricted to the cerebral cortex and may affect other grey matter regions, particularly the cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

A Rapid and Reliable Silver Impregnation Method forPneumocystis Cariniiand Fungi

AbstractA modified Grocott's methenamine-silver method for the rapid demonstration of Pneumocystis Carinii and fungi is described. Tissue sections are imbued in a dilute methenamine silver solution, then impregnated in a microwave oven, which is used as the initial heating source to promote rapid deposition of metallic silver in tissue structures. The sections are then transferred to a conventional oven to promote even, consistent, rapid, and reliable staining. (The J Histotechnol 11: 27, 1988.)

Parotid gland basement membrane variation in diabetes mellitus.

Post-mortem samples of parotid gland were obtained from 15 patients with a history of diabetes mellitus for a minimum of 5 years, and from 15 age- and sex-matched controls. The tissue was studied by direct immunofluorescence for abnormal binding of selected serum proteins, including IgG, IgM, IgA, C3, fibrinogen, polyvalent immunoglobulin and albumin, to acinar and ductal basement membranes of the gland. Thickness of these basement membranes was also assessed using a calibrated magnifier on uniformly enlarged photomicrographs of the tissue which had been stained by the chromotrope silver methenamine method to highlight basement membranes. Results of this investigation revealed parotid gland basement membrane abnormalities in all diabetic subjects as indicated by the binding of IgG, albumin and polyvalent immunoglobulins to ductal and acinar basement membranes. These basement membranes were uniformly negative in control subjects for the binding of all serum proteins tested. Binding of IgA was also noted in 7 of 15 experimental subjects, with 6 of these representing Type I diabetics. Basement membrane measurements revealed no difference in thickness between diabetic and non-diabetic subjects. Variations in parotid diabetic basement membranes evidenced in this study further substantiate the idea that membranopathy in this disease is systemic in nature.

Development of elastic fibers of nuchal ligament, aorta, and lung of fetal and postnatal sheep: an ultrastructural and electron microscopic immunohistochemical study.

The morphogenesis of elastic fibers of the nuchal ligament, aorta, and lung of sheep was studied by light microscopy, transmission electron microscopy, and immunohistochemical methods for the detection of elastin. The degree of maturation of the amorphous materials of elastic fibers was assessed morphologically in preparations stained by the tannic acid and periodic acid methenamine-silver methods. With both of these methods, the amorphous components of mature fibers stained less intensely than did those of immature fibers. Elastic fibers in early stages of development consisted of many microfibrils and few, small, branching masses of immature amorphous material. Thicker fibers were formed by the coalescence of growing masses of amorphous materials. In late stages of formation of elastic fibers, the mature amorphous materials were associated with few microfibrils; and they were partially surrounded by immature amorphous materials associated with many microfibrils. Antielastin antibody reacted evenly with amorphous materials in very early stages of elastic-fiber development, but reacted only with the other zones of amorphous materials in later stages; it also reacted with the microfibrils in all stages. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin on their surfaces. This conclusion is in agreement with ultrastructural observations showing 1) that development of microfibrils precedes that of the amorphous material and 2) that the microfibrils adjacent to the immature amorphous materials are covered with small amounts of tannic acid-positive amorphous materials. These observations suggest that microfibrils serve as sites for elastin deposition, both in early elastogenesis and in subsequent growth of elastic fibers. However, the nature of the interaction between elastin and microfibrils remains unknown.

The Ultrastructure of Carposporogenesis in Gigartina teedii (Roth) Lamour. (Gigartinales, Rhodophyceae): Auxiliary Cell, Cystocarpic Plant

Summary The auxiliary cell and the tissues of the female gametophyte (cystocarpic plant) have been studied during carposporogenesis in Gigartina teedii by light and electron microscopy and histochemistry. During the formation of gonimoblast cells, the auxiliary cell has an irregular shape and is multinucleated. It contains dense cytoplasm, few dictyosomes, numerous mitochondria, starch grains and plastids in a proplastid state. A covering layer of cells, derived from medullary filaments, is formed around the gonimoblast. It thickens and forms a medullary pericarp of haploid origin. The medullary pericarp cells are characterized by many conspieuous vacuoles (mucilage sacs), few dictyosomes, mitochondria and by plastids with a reduced thylakoidal system. These cells are also multinucleate. The inner medullary pericarp cells are less vacuolized, and are connected both with the gonimoblast cells and with peripheral elongated cells of the medullary pericarp. There are isodiametric cells with a rich, dense cytoplasm, numerous mitochondria and lipid droplets, microbodies and a few plastids with reduced thylakoidal structure. In addition there are elongate medullary pericarp cells between the gonimoblast cells. All these cells seem to serve as the nutritive tissue of the developing gonimoblast. During carposporogenesis the cortical cells surrounding the cystocarp divide repeatedly. The dictyosomes become hypertrophied and many vesicles containing polysaccharides (periodic acid silver-methenamine method) are formed from the Golgi apparatus. The wall of the epidermal cells does not give a positive PAS-reaction. This is in contrast to their apical cap formed by the contents of Golgi vesicles. The cell walls of the epidermal cells stains, however, with ruthenium red. The “cuticle” is about 5 μm thick and has a multilayered structure. The results of staining reactions suggest that it contains proteins and polysaccharides.

Staining of Fungi in Plastic-Embedded Tissue

Tissues embedded in plastic are sometimes stained with a special stain for fungi. Sections of plastic-embedded tissue were stained using both the periodic acid-Schiff (PAS) reaction and Grocott’s methenamine silver (GMS) method. The periodic acid-Schiff reaction is a more rapid and less complicated stain, which stained evenly. Grocott’s methenamine silver method stained inconsistently, occasionally caused sections to detach from the slides, and is a longer and more complicated stain. Thus, the periodic acid-Schiff reaction is recommended for demonstrating fungi in plasticembedded tissue.

Alterations in the distribution of glycoproteins in epithelial cells of murine colon after injection of tunicamycin

Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the change obtained by the silver methenamine method.

Cytochemical localization of complex carbohydrates in the thyroid gland of normal and TSH-treated mice.

The silver methenamine method for the ultrastructural localization of carbohydrates and glycoproteins was applied to the thyroid glands of normal and TSH-treated mice. The majority of the cisternae of the rough endoplasmic reticulum showed a weak, but apparently positive reaction. These findings support the opinion that glycosylation of thyroglobulin occurs initially in the rough endoplasmic reticulum. By this method the Golgi apparatus was observed to display a staining gradient. The intermediate to inner saccules were intensely stained, whereas the outer saccules were not so heavily stained. This phenomenon indicates that the Golgi apparatus has a functional polarity for the addition of carbohydrates to thyroglobulin and other proteins. In the inner and/or the peripheral regions of the Golgi apparatus and in the apical cytoplasm, a large number of globules of various sizes, considered to be colloid droplets, lysosomes and apical secreting vesicles, showed a positive reaction. The luminal colloid was also positive with silver methenamine staining, with almost the same intensity as the globules and vesicles.