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PubMedDate not supplied

Leishman-Giemsa cocktail: an effective Romanowsky stain for air-dried cytologic smears.

OBJECTIVE: To develop an economical, quick, readily available and simple alternative to May-Grünwald-Giemsa (MGG) stain and so to explore the combination of Leishman and Giemsa stain (LG cocktail). STUDY DESIGN: One wet-fixed and 2 air-dried smears were prepared from 720 cases during the period January 2003-November 2004. The LG cocktail and MGG stain were used on the air-dried smears and Papanicolaou stain in wet-fixed smears. Diagnoses of the cases were made using the LG cocktail-stained smears, and then its diagnostic efficacy was cross-checked with the MGG- and Papanicolaou-stained smears by the same cytopathologist. A comparative study of the LG cocktail and MGG-stained smears was done. RESULTS: The results achieved with the LG cocktail-stained smears were comparable to or even better than those with the MGG-stained smears, with excellent diagnostic efficacy. CONCLUSION: As compared to MGG stain, the 1-step LG cocktail is cheaper, easier to standardize and quicker.

Adenocarcinoma↗

PubMed1991

Staining of in vivo subsurface degradation in dental composites with silver nitrate.

A previously reported technique for staining areas of degradation in dental composite restorations was evaluated in 51 removed restorations. The staining reagent was silver nitrate, which penetrated the degraded subsurface as ionic silver and was subsequently developed into colored deposits of metallic silver. Several artefacts were recognized that resulted in an apparent image of subsurface stain. Most importantly, the presence of a layer of adsorbed silver on the edge of the specimen exaggerated the extent of staining. In order for the true depth of stain to be determined, thin sections of the materials should first be examined with a stereomicroscope to distinguish any contribution from adsorbed silver on the specimen edge. With this regimen, no stain was present in 41% of the restorations, and in a further 30%, the depth of stain was less than 50 microns. In two composites, the depth of stain was greater than 900 microns, and in a number of specimens, localized stain was found in association with attrition scars. Energy-dispersive x-ray analysis indicated that the amount of silver present in the degraded layers was very small. Overall, the results indicated that the staining technique is useful in the study of composite degradation.

Adsorption↗

PubMedDate not supplied

Integrated evaluation of central nervous system lesions: stains for neurons, astrocytes, and microglia reveal the spatial and temporal features of MK-801-induced neuronal necrosis in the rat cerebral cortex.

Routinely processed, hematoxylin and eosin (H&E)-stained slides are typically used to assess the morphologic integrity of the central nervous system in neurotoxicity safety studies. However, the value of special stains for improving neuropathologic evaluations during the assessment of neurotoxicity has been emphasized in the neuroscience literature and by regulatory agencies. The primary objective of the present study was to characterize the spatial and temporal changes in neurons, astrocytes, and microglia after dizocilpine maleate (MK-801)-induced focal neuronal necrosis in the posterior cingulate/retrosplenial (PC/RS) cortex of the rat. A secondary objective was to evaluate the application of special stains and a novel sectioning procedure for detecting neurotoxicity. Sixty adult male Sprague-Dawley rats were treated with sterile water vehicle or 10 mg/kg MK-801 and perfused through the left ventricle (pumped at 65 mm Hg pressure) with 10% neutral buffered formalin or 4% paraformaldehyde at 4 hr and on days 1, 3, 7, 14, and 28 after treatment. For light microscopic evaluation, brain sections were stained with H&E, a special cupric-silver (CS) stain that selectively impregnates degenerating neurons and makes them readily evident, glial fibrillary acidic protein (GFAP) immunohistochemistry for astrocytes, and Griffonia simplicifolia isolectin B4(GSA) histochemistry for microglia. Brains perfusion-fixed with 4% paraformaldehyde were prepared for CS staining with a novel frozen-sectioning procedure for multiple embedding in a composite gelatin block. In H&E sections from treated rats, necrotic nerve cell bodies were observed in PC/RS cortical layers 3 and 4 on days 1, 3, 7, and 14, but not on day 28. These necrotic neurons required high magnification for detection (x20 objective, x10 ocular). In contrast, degenerating neurons selectively stained with CS were observed in the same location as necrotic neurons seen with H&E but at low magnification (x2 objective, x10 ocular). Cupric-silver staining showed details not seen with H&E, including dendritic and axonal degeneration with progressive fragmentation. Beginning on day 3, GFAP immunohistochemistry revealed hypertrophic astrocytes in a diffuse pattern throughout the region of cell body necrosis, a change that persisted throughout the study. However, GSA lectin histochemistry identified a few reactive microglia on day 1 in a multifocal pattern throughout the region of cell body necrosis. Reactive microglia were observed on days 3, 7, and 14, but not on day 28. Glial changes observed with H&E staining were limited to an increase in the cellularity of glial cell nuclei in the area of neuronal necrosis. This study provides a comprehensive and integrated view of the temporal changes occurring in neurons, astrocytes, and microglia during acute neurotoxic injury. Moreover, advantages for using new staining and sectioning methodologies to enhance the toxicologic evaluation of the central nervous system are demonstrated.

Animals↗

PubMed1979

Interactions between pairs of DNA-specific fluorescent stains bound to mammalian cells.

The interactions between DNA-specific fluorescence stains complexed with mitotic Chinese hamster cells were studied by spectrofluorometric and flow fluorometric techniques. The degree of binding interactions and of energy transfer between stains was determined from the intensities and shapes of fluorescence emission spectra of cells complexed with pairs of stains. The stain pairs Hoechst 33258-chromomycin A3, Hoechst 33258-ethidium bromide, and chromomycin A3-ethidium bromide exhibited efficient energy transfer from the short wavelength absorber (donor) to the long wavelength absorber (acceptor), and little competitive or cooperative binding of stains. The stain pair quinacrine-ethidium bromide exhibited both energy transfer and competitive binding. None of the stain pairs showed evidence of strong electronic interactions between stains. The magnitude of energy transfer interactions was used to estimate the quantity and distribution of the stains molecules complexed to mitotic cells. The results indicate a fairly even distribution of each of these stains along the DNA of intracellular mitotic chromosomes.

Animals↗

PubMed1981

Postembedding staining of Brunner's gland with lectin-ferritin conjugates.

Postembedding staining of intracellular carbohydrates of rat Brunner's gland cells embedded in Epon and acrylamide was carried out with Ricinus communis agglutinin-ferritin, concanavalin A-ferritin, and wheat germ agglutinin-ferritin conjugates. Th Golgi vacuoles and mucous granules were stained with these conjugates. In each staining, the tissues embedded in acrylamide were stained more strongly than those embedded in Epon. The staining intensity of wheat germ agglutinin-ferritin was the strongest among the three conjugates and the staining intensity of Ricinus communis agglutinin-ferritin was stronger than that of concanavalin A-ferritin in both embedding methods. Free ferritin showed almost no binding to these structures and staining with the conjugates was inhibited by the addition of appropriate competitive sugars to the staining solutions. Osmium-postfixed tissues were not stained well with the conjugates. Washing of the sections with bovine serum albumin solution after staining was an essential step in the present method to reduce the nonspecific adsorption of the conjugates. The present method was very simple and had good reproducibility.

Animals↗

PubMed1982

Long-term storage and regular repeated use of diluted antisera in glass staining jars for increased sensitivity, reproducibility, and convenience of single- and two-color light microscopic immunocytochemistry.

A procedure is described for the dilution and storage of antisera in glass staining jars into which whole slides are immersed for incubation during light microscopic neuropeptide immunocytochemistry. Diluted antisera, stored at 4 degrees C and continuously reused, were found to be stable for long periods of time (to date over 3 years), and consistently yielded high quality staining in both single- and two-color immunoperoxidase staining. We found this procedure to be more convenient than conventional incubation procedures, allowing the more rapid processing of large numbers of slides and reducing the loss of slides due to technical errors. The consistency and reproducibility of day to day staining were also improved. The immersion of whole slides into the antisera permitted the use of long incubation times (up to 7 days) without the sections drying out, which in many cases substantially enhanced the sensitivity of the staining obtained. A procedure for two-color immunoperoxidase staining is described using diaminobenzidine for a brown color and alpha-naphthol/pyronin for a red/purple color. We found the alpha-naphthol/pyronin reaction superior to the more commonly used 4-chlornaphthol reaction as a second color. The two-color staining was found useful not only for demonstrating nerve cell bodies stained different colors, but also for staining nerve terminals one color that are around and contacting nerve cell bodies stained another color.

Adrenocorticotropic Hormone↗

PubMed1992

Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryofixed kidney and cartilage.

Cryofixation is generally believed to provide optimal tissue preservation. However, certain post-embedding cytochemical reactions, such as high-iron diamine (HID) staining for sulfated glycoconjugates, are not applicable to cryofixed and freeze-substituted tissues. In the present study, the HID technique was therefore adapted for post-embedding staining. HID staining was performed on thin sections of chemically and cryofixed kidney and growth plate cartilage, embedded in Epon and various acrylic-based resins. All resins and most tissue preparation conditions allowed post-embedding staining with HID, albeit to variable degrees. However, no significant cytochemical reaction was obtained with tissue sections of osmicated kidney embedded in Epon. Profile views of re-embedded sections showed that large stain deposits were usually restricted to the surface, whereas small ones were observed throughout the entire thickness of the section. The staining pattern was essentially similar between chemically fixed and cryofixed specimens. In the glomerulus, stain deposits were mainly seen over the free surface of podocyte foot processes and over the lamina rara externa. The pericellular cartilage matrix of chemically fixed specimens often appeared as condensed elements, usually stained with large deposits. In cryofixed tissues this matrix formed a meshwork composed of thin, extended filamentous structures, many of which showed linear arrays of smaller stain deposits. The data presented here indicate that post-embedding HID-TCH-SP staining can be successfully performed on thin sections of tissues embedded in various resins and, as a result, can be further adapted to cryo-prepared specimens to give a high resolution localization of sulfated glycoconjugates in tissues with optimal molecular preservation.

Animals↗

PubMed1992

Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows.

Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Backscattered electron imaging. Its application to biological specimens stained with heavy metals.

The present study demonstrates the applicability of backscattered electron images to the scanning electron microscope (SEM) observation of various biological specimens stained with heavy metals. Techniques of specimen preparation for backscatter imaging were introduced and results obtained by several staining methods were shown: silver staining for argyrophile fibers (Ishii's modification of Bielschowsky-Maresch's stain), Golgi staining for intracellular canaliculi of parietal cells, osmium-staining of intracellular lipid droplets, periodic acid-thiosemicarbazide-osmium staining for the detection of periodate-reactive mucosubstances and polysaccharides, and enzyme immunohistochemistry for bioactive peptides. Observation of stereo-pairs of backscatter images augments information on the three-dimensional localization of stained substances. Moreover, pairs of secondary emission and backscatter images of the same area clearly visualize the correlation between the surface structure and stained substances. Colored images by superimposition of secondary emission and backscatter information are also useful for an understanding of the orientation of the stained substances in three-dimensional structures. It is suggested that backscatter imaging can be widely used for various topochemical studies in three-dimensional extensions of biological specimens.

Adipose Tissue↗

PubMed1981

Factors affecting triple staining of human sperm.

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HCl (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.

Acrosome↗

PubMed1996

An automated double staining procedure for bone and cartilage.

Differential skeletal staining is an important part of developmental toxicologic studies. Traditionally these studies have required time-consuming differentiation of one or both stains used and careful attention to the maceration step to prevent specimen destruction. We present a fully automated protocol which does not require differentiation of either dye and incorporates a controlled maceration step which is highly reproducible. This has resulted in high quality staining that is reproducible, stable, and can be done in volume with minimal personnel time. The process involves the staining of skinned, eviscerated specimens fixed in 95% ethanol. Using an automated tissue processor, the specimen is stained in alcian blue for 24 hr, macerated in 3% potassium hydroxide for 24 hr and stained with murexide for 24 hr. The specimens are cleared and preserved in glycerol. Within three days specimens have red stained bone and blue stained cartilage. The procedure was developed using 20-day-old Sprague-Dawley rat fetuses to evaluate the feasibility of using the procedure for teratology studies involving the fetal skeleton. Evenly stained specimens can be examined within three days and stored for years without loss of staining.

Animals↗

PubMed1996

Highly selective acridine and ethidium staining of bacterial DNA and RNA.

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> or = 100 microM) of AO, lower concentrations (13.9 microM) of CPO, and did not stain with 0.5 microgram/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 microM) and CPO (13.9 microM) were shown to detect purified DNA in gels with a sensitivity in the range of 25-50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.

Acridine Orange↗

PubMed1996

Evaluation of preparation, staining and microscopic techniques for counting incremental lines in cementum of human teeth.

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.

Adolescent↗

PubMed1989

[Studies on human spermatozoal acrosome using spermac stain in fertile and infertile men including two cases of round-headed spermatozoa].

Using a new acrosome stain named "Spermac stain", the relationship between the staining rate of sperm acrosome and conventional semen parameters, i.e., sp. density and sp. motility efficiency index (SMEI) was studied. The mean positive staining rate of sp. acrosome in ten fertile men was 81.5%. In 72 infertile men, a significantly higher (p less than 0.001) correlation was noted between the positive staining rate and sp. density and SMEI. 5 out of 8 male partners of unexplained infertility couples showed low staining rates (40-60%), in spite of normal sp. density and SMEI. Ejaculates from two infertile men with "round-headed" spermatozoa were examined by Spermac stain and electron microscopy. The staining rates of sp. acrosome in these two cases were 4.5% and 16.5%. The fine structures of the sperm and the spermatid were presented. Because Spermac stain is quick, reliable and easy to use, this staining method is recommended for evaluating sp. acrosomal morphology in practical use.

Adult↗

PubMed2001

Uveal melanoma: mean of the longest nucleoli measured on silver-stained sections.

PURPOSE: To summarize and compare the various histologic methodologies for using nucleoli to assess the malignant potential of uveal melanoma. METHODS: This was an observational series of 100 samples of uveal melanoma in which histologic sections were studied. The cases were selected so that approximately half (n = 49) of the tumors were from patients who had died of metastatic malignant melanoma. The 51 remaining tumors were from patients who had survived at least 9 years without development of metastasis. Central sections from the uveal melanomas were stained using the colloidal silver nitrate stain for nucleolar organizing regions (AgNOR). These were compared with an adjacent hematoxylin and eosin (H&E)-stained section. A light microscope with a micrometer inset into the eyepiece (x10) was used at a final magnification of x1000 under oil immersion to measure the length of the nucleolus along the longest axis and the width perpendicular to that axis. From at least twenty cells selected from random fields throughout the tumor, the mean of the 10 longest and widest nucleoli (MLN) was calculated. Seven samples had to be discarded because the nucleoli were unmeasurable. RESULTS: T-tests and Cox proportional hazard regression analysis indicated that the MLN of nucleolar length as measured on AgNOR-stained slides was as significant as cell type but was more significant than other histopathologic prognosticating variables measured and evaluated in this study. These prognosticators included tumor size, calculated as the largest tumor dimension; MLN width; and MLN length, as measured on H&E-stained sections. CONCLUSIONS: It has previously been demonstrated that AgNOR-stained nucleoli, unlike H&E-stained nucleoli, can be captured and measured by an automated image analyzer with prognostically significant results. This new method of simple oil-immersion measurements of the longest AgNOR-stained nucleoli length in microscopic sections of uveal melanoma provides an inexpensive and highly significant method for predicting outcome in patients with uveal melanoma. Because of the high contrast with the background, the silver-stained nucleoli clearly define the nucleolar boundaries, rendering them readily discernible and allowing greater ease and speed of measurement when compared with H&E-stained nucleoli. The method of random sampling that was used was comparable with linear sampling in predicting outcome. Highly necrotic tumors, however, had to be excluded from the study because of loss of nucleolar morphology.

Cell Nucleolus↗

PubMed1999

Diagnosis of Blastocystis hominis by different staining techniques.

One hundred and fifty stool samples were collected from diarrheic patients of different ages, and examined for Blastocystis hominis by direct smears and concentrated by Sheather's sugar flotation. Staining was done by: Giemsa, two modifications of trichrome stain, modified Ziehl-Neelsen, safranin-methylene blue and two-auramine stains. Out of the 150 cases nine were positive for blastocystosis. The best stains were safranin-methylene blue and modified Ziehl-Neelsen stains. They had the advantage of staining cysts and amoeboid forms besides being rapid and easy to perform. The modified trichrome stains identified 8 ie, less specific and were time consuming. The auramine dyes stained the cyst, both the wall and internal body fluoresced brightly. Giemsa stain was not an efficient stain. Scanning and transmission electron microscopy (SEM, TEM) were performed to study the fine ultrastructure.

Animals↗

PubMed2003

[Improvement of the silver-stain and it's test and verification in comet assay].

In order to find out a better silver-stain method so that to replace EB-stain in comet assay, the selected silver-stain methods were tested via determining fixing, coloring, displaying, fading, and testing the pigmentation temperature. When it was optimized, it was used in testing the DNA damage by two types of compounds, namely potassium dichromate (K2Cr2O7) and 1,2-dichloroethane (DCE) to two kind of cells, namely spleen cells of mouse and human outer lymphocyte, and, the sensitivity was compared with that of EB-stain. The results showed that the modified silver-stain was the first successful non-fluorescent dye method in comet assay that can dye clearly, permanently, with a light background and good contrast, and it was easy to manipulate and it was cheap. The stain can detect the DNA damage by compounds to cells and has the same positive threshold like that in EB-stain. It can even dye the small fragments that EB-stain cannot. It is suggested that in comet assay, the silver-stain can substitute the EB-stain completely.

Comet Assay↗

PubMed2004

[Effect of staining method and sintering temperature on the color of porcelain-fused-to-metal restorations].

PURPOSE: To investigate the effect of staining method and sintering temperature on the color of porcelain-fused-metal restorations. METHODS: 40 cylindrical stained porcelain-metal specimens of 15 mm in diameter and 6 mm in height were fabricated with customized mould, consisting of 2 mm Ni-Cr metal, 1 mm opaque porcelain, 2 mm dentine porcelain and 1 mm enamel porcelain. The specimens were prepared by 5 techniques, 8 for each group. Group A: internal staining, Group B: external staining, 900 degrees centigrade sintering temperature was used in both A and B group; Group C to E: external staining, with the sintering temperature of 880 degrees centigrade, 900 degrees centigrade and 920 degrees centigrade respectively. Sofu A2 porcelain and Sofu 44 stain system were used for the study. Using standard white plate as a reference, colors (L*,a*,b* coordinates) of the specimens were measured with a computerized colorimeter. Student's t test and one way ANOVA were used to analyze the data. RESULTS: DeltaE, b* and Delta C(ab)* of Group B (external staining) and group A (internal staining) were 43.72 +/- 2.99/26.51 +/- 1.64/31.31 +/- 2.48 and 39.71 +/- 1.78/23.69 +/- 0.36/26.55 +/- 2.16, respectively. The values of the former group were significantly higher than that of the latter (P < 0.05); For Group C to E, there were no significant differences in all the color parameters. CONCLUSIONS: Staining method has a significant effect on the color parameters of porcelain-fused-to-metal restorations; For external staining, within the clinically-used range, changing the sintering temperature does not have an obvious effect on the color.

Color↗