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PubMed1961

Preferential staining of nucleic acid-containing structures for electron microscopy.

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10(-2)M Ca(++), and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0 degrees C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times ((1/2) hour to 2 hours, at 0 degrees C or 20 degrees C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.

Coloring Agents↗

PubMed1996

Oviductal protein produces fluorescence staining of the perivitelline space in mouse oocytes.

Mouse oocytes were previously observed to undergo structural changes involving the perivitelline space (PVS) within the oviduct following ovulation, as visualized by staining with fluorochrome-protein conjugates. In the present study, this phenomenon was investigated in detail to determine the role of the oviduct and oocyte. Mouse ovarian oocytes matured in vitro were further incubated in medium or within explanted oviducts in vitro for varying periods of time and then stained with fluorescein isothiocyanate (FITC)-casein. Twenty percent of oocytes incubated within explanted oviducts for 3 hr showed distinct fluorescence staining of the PVS, whereas after 20 hr incubation, most (89%) oocytes were similarly stained. In contrast, no ovarian oocytes was stained when incubated in medium alone. Puromycin treatment during incubation of oocytes within explanted oviducts produced a dose-dependent decrease in the percentage of oocyte exhibiting PVS staining after FITC-casein exposure. FITC-casein staining of the PVS also occurred in all oocytes following incubation of in vitro-matured oocytes with oviductal tissue extract. In contrast, no oocytes incubated with serum exhibited fluorescence staining. Additionally, the PVS of oocytes failed to stain after incubation with either 0.001% of trypsin- or heat-treated oviductal homogenate. When zona pellucida (ZP) ghosts, devoid of ooplasm, were incubated within explanted oviducts, their PVS was stained brightly following FITC-casein treatment. From these results, it is concluded that proteinaceous material(s) secreted by the mouse oviduct is responsible for the fluorescence staining of the PVS of mouse oocytes and of ghost ZP. The ooplasm does not appear to play any role in altering the properties of the PVS staining.

Animals↗

PubMed2000

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain.

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.

Amino Acid Sequence↗

PubMed2006

Nucleolar staining cannot be used as a screening test for the scleroderma marker anti-RNA polymerase I/III antibodies.

OBJECTIVE: Anti-RNA polymerase I/III (anti-RNAP I/III) antibodies are clinically useful markers of scleroderma, and their presence is associated with diffuse skin disease and an increased risk of cardiac and kidney involvement. Although RNAP I antibodies localize to the nucleolus, nucleolar staining by many anti-RNAP antibody-positive sera is not always observed. Nucleolar staining by anti-RNAP antibody-positive sera was examined by double staining with antifibrillarin antibodies to evaluate whether nucleolar staining can be used as a screening test for anti-RNAP I/III antibodies. In addition, the relationships between nucleolar staining and levels of anti-RNAP III antibodies were examined by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP) assay. METHODS: Sera were tested using immunofluorescent antinuclear antibodies on HEp-2 cell slides, by anti-RNAP III ELISA, and by IP assay using (35)S-labeled K562 cell extract. Nucleolar staining by anti-RNAP antibody IP-positive sera was confirmed by double staining using antifibrillarin monoclonal antibodies. The levels of anti-RNAP III antibodies were quantitated by ELISA and by IP assay using a serially diluted reference serum as a standard, and their relationship was analyzed. RESULTS: All 18 anti-RNAP I/III antibody-positive sera showed nuclear speckled patterns, but nucleolar staining was readily noticeable in only 44% of the sera. A positive correlation was found between ELISA and IP units for anti-RNAP III antibodies. The levels of anti-RNAP III antibodies and anti-RNAP I antibodies correlated well, with the exception of a few sera. Levels of anti-RNAP III antibodies were low in sera with nucleolar staining, whereas several sera with high levels of anti-RNAP I antibodies clearly showed nucleolar staining. CONCLUSION: Although some sera positive for anti-RNAP I/III antibodies clearly stain nucleoli, nucleolar staining is inconsistent and cannot be used to screen for anti-RNAP I/III antibodies.

Autoantibodies↗

PubMed1997

Computer analysis of corneal innervation density using a novel double stain in rat corneal whole mounts.

Computerised morphometry and a double stain technique were utilised to examine the corneal nerves in whole mounts. This novel stain combines the nonspecific acetylcholinesterase (NsAchE) and gold chloride (AuCl) procedures to enhance staining contrast and facilitate computerised detection of corneal nerves. Fresh rat corneas were dissected, and the Descemet's membrane-endothelium complex was removed. Then the corneas were fixed in 4% paraformaldehyde with 50 mM Na-K phosphate buffer (pH 7.2) and 8% sucrose for 30 min. They were rinsed and stained singly with NsAchE or AuCl, or were double stained using NsAchE followed by AuCl. Between NsAchE and AuCl staining the corneas were stored frozen in OCT compound at -70 degrees C. Flat mounts of whole corneas were photographed before and after the second staining. Measurable stromal innervation density (mean +/- S.D.) in age-matched corneas stained with AuCl (3.90 +/- 0.36 mm/mm2) was not significantly different from that of NsAchE stained corneas. However, double staining compared with NsAchE staining of the same corneas revealed a 48 +/- 27% increase in demonstrable innervation density of the subepithelial nerve plexus (7.95 +/- 0.86 mm/mm2 vs 5.52 +/- 1.31 mm/mm2, respectively). Improved visualisation of epithelial nerves and their fine ramifications (leashes) was also obtained by double staining. This novel combination of 2 procedures enhances the detection of corneal nerves for analysis by computerised morphometry and provides a more representative estimate of total corneal innervation density.

Acetylcholinesterase↗

PubMed1982

Relationship between fluorescein diacetate-stained hyphae and oxygen utilization, glucose utilization, and biomass of submerged fungal batch cultures.

The relationship between fungal activity and staining with fluorescein diacetate (FDA) was investigated by growing Penicillium citrinum and Rhizoctonia solani in submerged batch cultures at different initial glucose concentrations and aeration rates. A modified FDA staining method, similar to the Jones and Mollison technique (P. Jones and J. Mollison, J. Gen. Microbiol. 2:54-69, 1948), was developed to assess both total and FDA-stained hyphae. In previous studies, soil hyphae stained with FDA were considered viable. However, determination of a quantitative relationship between FDA staining and fungal activity is necessary before such an assumption can be made. Growth rates and the rate of change in the percentage of FDA-stained hyphae were significantly correlated. The regression equation calculated for the relationship was: growth rate (mg . ml-1 . h-1) = 0.34 + 1.1 (rate of change in the percentage of FDA-stained hyphae [. ml-1 . h-1]). Changes in activity as measured by O2 utilization, glucose utilization, and biomass correlated significantly with changes in the percentage of FDA-stained hyphae, although the relationships among these parameters were different for each fungal species. Fungal growth stage was also correlated with the percentage of FDA-stained hyphae. Staining was 10% or greater during fungal growth and less than 10% during the late growth, stationary, and death phases. Thus, the rate of change in the percentage of FDA-stained hyphae can be used to predict fungal activity rate changes for single fungal cultures and growth rates for mixed fungal cultures, and the growth stage can be assessed by the percentage of FDA-stained hyphae.

Aerobiosis↗

PubMed1979

Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology.

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.

Animals↗

PubMed1984

Species-specific second antibodies reduce spurious staining in immunocytochemistry.

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.

Animals↗

PubMed2002

Extrinsic stain removal with a sodium hexametaphosphate-containing dentifrice: comparisons to marketed controls.

Repeated exposure to chlorhexidine and tea has long been recognized to promote extrinsic tooth stain. Previously, the merits of a modified, rapid clinical trial model using chlorhexidine and tea in combination with restricted brushing to accelerate staining have been established. A nine-week randomized and controlled, examiner blind, parallel group clinical trial was conducted to compare selected chemical and mechanical approaches for stain removal using this model. After prophylaxis, stain was induced during a 3-week pre-treatment period, and then the healthy adult volunteers were randomized to one of three treatment groups: an experimental whitening dentifrice containing a combination of sodium hexametaphosphate and soluble pyrophosphate; a marketed alumina-based high abrasive dentifrice; or a lower abrasive control dentifrice. At baseline, three and six weeks, dental stain was measured on the facial and lingual surfaces of the eight anterior teeth using the Lobene index. A total of 94 subjects completed the study. Both the chemical and abrasive dentifrices showed statistically significant reductions in stain area (p < 0.05) relative to the control after three weeks. After six weeks, adjusted mean stain area scores were 0.35, 0.41 and 0.49 for the sodium hexametaphosphate, alumina and regular dentifrice groups, respectively. While the alumina dentifrice showed only a directional benefit, the sodium hexametaphosphate differed statistically (p = 0.023) from the control. Subjects in both the chemical and abrasive dentifrice groups showed statistically significant reductions relative to baseline for Lobene composite stain, stain area and stain intensity (p < 0.05) at six weeks. There were no statistically significant differences in stain levels between the sodium hexametaphosphate and alumina dentifrice groups at any time. This study demonstrates that following six weeks of treatment, sodium hexametaphosphate-containing dentifrices are effective in removing extrinsic tooth stain, with performance at least comparable to that seen with more abrasive dentifrices.

Adult↗

PubMedDate not supplied

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues.

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G<acid Fuchsin<Light green<Methyl Blue<Fast Green), which are used simultaneously, and it enables the individual tissues to be selectively differentiated and stained. Muscle fibers, collagen, reticulin and elastin fibers, erythrocytes, cartilage, bone, mucous cells, oocytes and larvae were selectively stained and differentiated. Dyes with small size and molecular weight (i.e Orange G), penetrate all tissue structures rapidly, but are only tightly retained in densely textured tissues (i.e erythrocytes). Methyl Blue is an interesting triarylmethane dye (large size and molecular weight), which is incorporated in this new VOF tetrachrome stain, and acquires histochemical significance when used at acid pH (2.8) because collagen and reticulin fibers, as well basophilic and metachromatic substances (strongly ionized sulphated glycoconjugates) can be identified. Muscle tissues show an evident green colour (Fast Green or Light Green affinities), even those isolated and/or diffuse muscle fibers present in the digestive submucosa layer. Connective tissues showed a specific and strong blue colour (Methyl Blue affinity) or mixed blue-red staining (Methyl Blue and Acid Fucshin affinities). Very noticeable is the staining of the mucous cells, as well as the hyaline capsule of the viral lymphocystic cells, which were stained blue-purple (carboxylated and/or strongly ionized sulphated groups). Cartilaginous tissues showed a blue or purple (Methyl Blue affinity) staining, and a specific red colour (Acid Fucshin affinity) was evident during calcification or in bone structures (i.e skeleton, fins, gills, teeth).

Animals↗

PubMed1991

Location of port-wine stains and the likelihood of ophthalmic and/or central nervous system complications.

Of 310 patients with port-wine stains, 68% had more than one dermatome involved; 85% had unilateral and 15% had a bilateral distribution of their port-wine stain. At the time of examination, 8% of all patients with trigeminal port-wine stains had evidence of eye and/or central nervous system (CNS) involvement. Extensive involvement, with port-wine stain over the trunk and extremities as well as the head and neck, was observed in 12%. Patients who did not have port-wine stains on the areas served by branches V1 and V2 of the trigeminal nerve had no signs or symptoms of eye and/or CNS involvement. Port-wine stains of the eyelids, bilateral distribution of the birthmark, and unilateral port-wine stains involving all three branches of the trigeminal nerve were associated with a significantly higher likelihood of having eye and/or CNS complications. Twenty-four percent of those with bilateral trigeminal nerve port-wine stains had eye and/or CNS involvement compared with 6% of those with unilateral lesions. All those who had eye and/or CNS complications had port-wine stain involvement of the eyelids; in 91% both upper and lower eyelids were involved, whereas in 9% only the lower eyelid was involved. None of those with upper eyelid port-wine stains alone had eye and/or CNS complications. In addition, 3 (75%) of the 4 subjects with seizures alone had bilateral port-wine stain involvement. A third group, these with unilateral V1, V2, and V3 port-wine stains, had eye and/or CNS complications in 3 (19%) of 16 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗

PubMed1994

Assessment of immunocytochemical and histochemical stainings in the distinction between reactive mesothelial cells and adenocarcinoma cells in body effusions.

BACKGROUND: The accumulation of fluid in body cavities is a common event in both neoplastic and non-neoplastic diseases. However, the distinction between cells of reactive process and those of malignancy in cytology is not always possible. It is especially difficult when reactive mesothelial cells and adenocarcinoma cells are encountered. The aim of the present study was to find out the most accurate or reliable immunocytochemical and histochemical stains to distinguish reactive mesothelial cells from adenocarcinoma cells, and to serve as a standard method in the future when dealing with equivocal cases. METHODS: Ninety-nine cases of malignant epithelial effusion were collected from 755 cases of effusion obtained from 3 large body cavities in the past one-year period. Among them, 71 cases were histologically as adenocarcinoma and 13 cases as non-adenocarcinoma. The other 15 cases were carcinoma proved by image as well as clinical symptoms and signs. These 99 cases, plus 10 cases of non-malignant effusion, underwent immunocytochemical and histochemical stainings. Five common commercial antibodies used in this immunocytochemical study were epithelial membrane antigen (EMA), carcinoembryonal antigen (CEA), cytokeratin, vimentin and Leu-M1. The histochemical study included periodic acid-Schiff diastase (D-PAS) and mucicarmine stains. RESULTS: The immunocytochemical study showed that EMA had a high frequency of positive staining with malignant epithelial cells and a negative staining with mesothelial cells. Cytokeratin always stained with malignant epithelial cells but it also stained with mesothelial cells. Almost all the proved adenocarcinomas expressed CEA which was not expressed in the proved non-adenocarcinomas. However, CEA occasionally stained mesothelial cells. Leu-M1 showed a low frequency of staining with the proved adenocarcinoma cells, but it did not stain proved non-adenocarcinoma cells and mesothelial cells. On the contrary, vimentin stained with all mesothelial cells but occasionally with malignant epithelial cells, especially the proved adenocarcinoma cells. In the histochemical study, both mucicarmine and D-PAS showed a low sensitivity but high specificity in detecting the adenocarcinoma cells. CONCLUSIONS: No single marker is absolutely reliable to distinguish exfoliated, reactive mesothelial cells from adenocarcinoma cells in effusions. However, a panel of 3 antibodies containing EMA, CEA and vimentin, together with D-PAS and mucicarmine stains may help solve this problem.

Adenocarcinoma↗

PubMed1993

[Study on the color modification buring surface staining of PLAT castable ceramic restorations].

The surface staining of castable ceramic restorations is a technique-dependent process. So it is inevitable that the color of restorations mismatch the color of natural teeth. This study was conducted to explore the possibility of modifying the color by selected use of special colored stains. Eighty PLAT castable ceramic samples were machined, 16 mm in diameter, 1.2 mm in thickness. Thirty-two samples were stained with yellow stain and 32 samples orange. Sixteen samples were stained with red, green, blue and violet stains, respectively. To the yellow and orange stained samples, we applied correspondingly the same stain, red, green, blue and violet stains. The color of each sample was evaluated with a colorimeter and translated to Munsell notations. The results indicated that chroma was intensified by each application of the same stain. Red and green stains made the hue reddish and greenish. Blue and violet stains could effectively decrease the chroma of yellow or orange. Meanwhile, the hue was also slightly changed to greenish and reddish.

Ceramics↗

PubMed1995

A comparative study of stain removal with two electric toothbrushes and a manual brush.

Recent studies have suggested that a sonic electric toothbrush is more effective than a manual brush at removing extrinsic dental stain. There have been few studies of the comparative stain removal properties of different electric brushes. The study reported here was conducted to compare the efficacy of the sonic toothbrush (Sonicare) with an oscillating/rotating brush (Braun Oral-B Plaque Remover) and a conventional manual brush (Crest Complete). The study was a single-blind, randomized, cross-over design, balanced for residual effects and employing 24 subjects. Stain was enhanced over a 21-day period by twice-daily rinses with chlorhexidine and frequent intakes of tea and/or coffee. At the end of each period, tooth stain intensity and area, tongue stain intensity and area, lower lingual calculus and subjective tooth sensitivity were recorded together with preference for the brushes determined at the study's completion. Similar levels of tongue staining were recorded for the three periods, with no significant differences between the three groups. Tooth stain intensity, for most sites, was not significantly different between the three groups. For mean total stain area and for lingual and lingual interproximal sites, a significant reduction in stain was seen following use of the oscillating/rotating brush compared to the manual brush. The reductions in stain with the sonic brush were not significantly different from the manual brush. With the exception of maximum stain intensity, there were no significant differences between the oscillating/rotating and sonic brushes. Significantly less tooth sensitivity was found following use of the oscillating/rotating brush compared to both the manual and sonic brushes. All three brushes were found to be safe, but volunteer preference significantly and predominantly favored the oscillating/rotating brush. The results suggest that the oscillating/rotating brush is superior to a manual brush for stain removal.

Adult↗

PubMedDate not supplied

Modified Giemsa staining for rapid diagnosis of malaria infection.

OBJECTIVES: To develop and evaluate a rapid method for the diagnosis of malaria infection by microscopy of stained blood films. SUBJECTS AND METHODS: Blood specimens were collected from randomly selected confirmed malaria cases (n = 75) and suspected malaria cases (n = 175). The microscopy was done on each set of blood films stained by modified and the standard Giemsa staining methods. RESULTS: All the 75 previously diagnosed malaria cases were confirmed by the microscopy of blood films stained by both methods. Forty-nine (28%) of the 175 cases suspected for malaria infection showed malarial parasites on microscopy of blood films stained by both methods. However, due to homogeneous staining and clearer background of the blood films it was possible to determine the parasite species in 65% of the cases on microscopy of the thick films stained with the modified method compared to only 20% with the standard method. Further, the turnaround time for reporting the microscopy test result was 15-20 and 45-50 min with modified and standard staining methods, respectively. CONCLUSION: Our data showed that performance of the modified staining method in detecting malarial parasites was comparable to that of the standard staining method. Moreover, the modified staining method was rapid, easy to use, and reliable.

Animals↗

PubMed1975

The use of a basic dye (azure A or toluidine blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study.

Selective purple staining of RNA-rich structures such as basophilic cytoplasms of exocrine pancreas and plasma cells, Nissl substance, and nucleoli was achieved by treating tissue sections as follows. Stain dewaxed sections for 1/2 hour in a dyebath containing 0.1% w/v axure A or toluidine blue and 1% cationic surfactant (Hyamine 2389, a 50% w/v aqueous solution of diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride; or benzyldimethylammonium chloride, or cetylpyridinium bromide, or cetyltrimethylammonium bromide) buffered to pH 7 with phosphate. Rinse in water, blot, air dry and mount in synthetic resin. Intense purple staining of RNA-rich regions occurred after fixation in neutral formalin or in Carnoy's or Gendre's fluids, though satisfactory results were also found after fixation in acetone or alcohol. Chromatin generally stained a very pale azure after all fixations, though occasionally nuclei were unstained (Gendre's or Zenker's fluids). Subjecting tissue sections to acid hydrolysis or to digestion by RNAase eliminated or reduced the purple staining, but left the azure staining of nuclei unaffected. Satisfactory staining of RNA-rich structures was not critically dependent on the precise concentrations of dye, surfactant or inorganic salts in the dyebath, nor on pH, staining time or chemical nature of the surfactant. The staining patterns can be rationalized with a tissue model that considers both surface charge and permeability factors, since present in the dyebath are small dye cations and large cationic surfactant micelles. As micelles and dye will both quickly penetrate basophilic structures considered to be porous, such as chromatin, competition will then greatly reduce staining of such substrates. But the large micelles will only slowly penetrate regions considered to be more impermeable, such as basophilic cytoplasms, so consequently small fast moving dye ions may enter and stain without competition.

Animals↗

PubMed1988

Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density.

To determine whether accurate quantification of Pneumocystis carinii cysts in sputum is possible using currently available means and to determine the optimal stain for screening sputum for P carinii, we undertook a comparative evaluation of readily available stains: (1) Diff-Quik, a stain that provides tinctorial detail comparable with Giemsa, (2) toluidine blue, and (3) silver methenamine on simultaneously thawed sputum samples containing P carinii. We also studied the Diff-Quik stain in combination with (4) toluidine blue and (5) silver methenamine. The three stains were comparable for screening purposes. Each disclosed similar numbers of P carinii aggregates on similarly prepared slides. Cysts were difficult to recognize on Diff-Quik due to the negatively staining character of the cyst wall with this stain. The cyst-wall stains, toluidine blue and silver methenamine, clearly stained the cyst wall. The combination of a cyst-wall stain with Diff-Quik allowed visualization of cysts, highlighted by the cyst-wall stain, and aggregate area, delineated by the Diff-Quik, allowing expression of number of cysts per aggregate area, a measure termed cyst density.

Animals↗

PubMed2001

Practical aspects of fluorescent staining for proteomic applications.

SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5,000 with washing solution for 3 or more hours, produced high sensitivity, low background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.

Electrophoresis↗