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PubMed1984

[Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect].

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

Prospective comparison of H&E, Giemsa, and Genta stains for the diagnosis of Helicobacter pylori.

BACKGROUND: H. pylori is more easily visualized with special stains than with H&E, but this adds time and expense to the diagnostic workup. We sought to determine if the diagnostic accuracy was improved with special stains. METHODS: One hundred-one patients had two "jumbo" biopsies taken from the gastric antrum and two from the body for examination with H&E, Genta, and Giemsa stains. Four separate biopsy specimens were also taken from the antrum and the body for culture and for three types of rapid urease test, and 13C-urea breath tests were also performed. Mixed, coded biopsies were assessed for H. pylori, and density was scored from 0 to 4. A case was considered positive for H. pylori if culture was positive, two rapid urease tests and a urea breath test were positive, or two different stains were positive. Biopsy specimens were excluded from analysis if the slides were missing or there was inadequate tissue for review, or if the specimen showed a lack of staining. RESULTS: Fifty-two (13%) of 404 specimens were excluded because of a poor Genta stain. Sensitivities were comparable for the three stains (H&E, 92%; Giemsa, 88%; Genta, 91%), while H&E specificity (89%) was significantly lower than that of the special stains (98%). Sensitivity for all three stains was significantly lower at low (grade 0 to 1) H. pylori density than at high (grade 2 to 4) density (H&E, 70% vs 98%; Giemsa, 64% vs 96%; Genta, 66% vs 97%), and 20 of 22 false positives were grade 1. CONCLUSIONS: The sensitivities of H&E and special stains are comparable at around 90%, but the specificity of H&E is significantly lower. The Giemsa stain appears to be the preferred stain for H. pylori diagnosis on the basis of its good sensitivity, excellent specificity, and lack of technical difficulty in preparation. However, H&E provides excellent accuracy when more than minimal (grade 1) H. pylori density is present.

Adult↗

PubMed2006

Application of modified ultrafast Papanicolaou stain in cytology of various organs.

This prospective study was conducted to assess the use of modified ultrafast Papanicolaou (MUFP) stain for fine-needle aspiration cytology (FNAC) of various organs. In India, many reagents are not easily available. In this modification, Gill's haematoxylin, EA-36, and isopropanol substitute Richard-Allan haematoxylin, cytostain, and ethanol, respectively. Group I included 40 FNAC smears of various organs. In each case, three smears were prepared and stained by MUFP, Papanicolaou, and May-Grunwald Giemsa (MGG) stains. In group II, 10 intraoperative cytology smears of different organs were stained with MUFP and rapid haematoxylin-eosin (H&E). For assessment of MUFP stain, scores were given on four parameters: background of smears, overall staining pattern, cell morphology, and nuclear staining. Quality index was calculated from ratio of score achieved to maximum possible score. Diagnosis made by MUFP stain was compared with standard stains. It was observed that MUFP-stained smears had clear RBC free background, crisp nuclear chromatin, well-stained nucleoli, and transparent cytoplasm. The diagnosis made was correct except in three cases of metastatic squamous-cell carcinoma. Hence, it was concluded that MUFP stain is useful for rapid diagnosis by FNAC, but is not useful for squamous-cell lesions.

Breast Neoplasms↗

PubMed2006

Utility of reflex Gomori methenamine silver staining for Pneumocystis jirovecii on bronchoalveolar lavage cytologic specimens: a review.

Pneumocystis jiroveci (Pj; formerly Pneumocystis carinii) is an opportunistic pathogen causing life-threatening pneumonia (Pneumocystis pneumonia) in immunosuppressed individuals. Its diagnosis is dependent on identification in bronchoalveolar lavage (BAL) specimens. Gomori's methenamine silver nitrate (GMS) stain has been advocated to highlight the organisms in BAL specimens. This study was performed to determine the utility of reflex GMS staining on all BAL specimens for identifying Pj.All BAL specimens from years 2000 to 2004 were processed as cytospins and stained with Papanicolaou (Pap) and GMS stains. A total of 2,984 BAL specimens were identified. A total of 116 (3.9% of total BAL) BAL specimens were diagnostic of Pj. The diagnostic specimens were grouped as follows: 103 (88.8% of total positive cases) Pj identified with both Pap and GMS staining; 11 (9.5% of total positive cases) Pj identified only with Pap staining; and 2 (1.7% of total positive cases) Pj identified only with GMS staining. In conclusion, the prevalence of Pj in BAL specimens is 3.9%, which can be attributed to improved management of immunocompromised patients. Performing reflex GMS staining on all BAL specimens does not improve the diagnostic identification of Pj since the majority (98.3%) of diagnoses can be rendered on Pap stained slides. A cost analysis for GMS staining on 2,879 GMS-negative BAL specimens was estimated at $143,950. Thus, from diagnostic and cost benefit perspectives, GMS staining can be recommended only on cases where Pap stain is negative, and the clinical presentation is consistent with Pneumocystis pneumonia.

Adolescent↗

PubMed1994

In vivo staining of oligodendroglia in the rabbit retina.

We have discovered that a strongly fluorescent dye, sulforhodamine 101, when injected intravitreally in vivo, very effectively stains a class of star-shaped cells in the innermost layers of the rabbit retina. The cells were strictly confined to the region containing medullated fibers and emitted dichotomously branching processes that ended up running some distance along the myelinated fibers. In favorable cases they could be seen to ensheath the fibers in a tube-like fashion. No other retinal cells were stained. Shortly (hours) after the injection, the stain appeared in the cell cytoplasm, but it later became progressively more localized to intracellular granules. Most of the dye had disappeared after 2 days. Oligodendrocytes and astrocytes are the only cells known to be confined to the region of the medullated fibers in the rabbit retina, and hence the sulforhodamine 101-stained cells should be one of these two types. Sulforhodamine 101-stained cells were indistinguishable from oligodendrocytes identified by 2',3'-cyclic nucleotide phosphodiesterase (CNP) immunohistochemistry, and sequential staining showed them to be the same. Sulforhodamine 101-stained cells were microinjected with lucifer yellow after lightly fixing rabbit retinas with formaldehyde and were found to be indistinguishable from oligodendrocytes. Glial fibrillary acidic protein staining for astrocytes showed fiber bundles that to some extent were similar to the bundles stained by sulforhodamine 101, but at the level of individual fibers, it was impossible to establish any concordance. Sulforhodamine 101 thus appears to stain oligodendrocytes rather than astrocytes in the rabbit retina. A related dye, rhodamine 123, also stained rabbit oligodendrocytes, but with poor contrast because many other cells and structures were also stained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980

Lectin staining of carbohydrates of haemic cells; the cells of normal blood and bone marrow and of the myeloid leukaemias.

Plant proteins and aprotinin (a protein of beef lung), labelled with fluorescein isothiocyanate, were used as histochemical tools for the demonstration of carbohydrates. Sialic acid (or glucuronate) was stained with aprotinin (FLA); galactose was stained with Ricinus communis agglutinin (FL-RCA) and mannose (or glucose) with Concanavalin A (FL-Con-A). Normal human bone marrow and blood were examined as were the cells of patients with acute and chronic myelogenous leukaemia. The plasmalemma, cytoplasm and nuclear membrane of the cells of the normal granulocytic series were stained well with FLA, but the corresponding leukaemic cells fluoresced less intensely. Chromatin was weakly stained in both normal and leukaemic cells. FLA-RCA and FL-Con A stained the plasmalemma, cytoplasm and nuclear membrane weakly, but did not demonstrate chromatin. There was no detectable difference between normal and leukaemic cells. Eosinophil and basophil granules--in contrast to those of the neutrophils--stained well with all three compounds, in both the normal and leukaemic cells. In megakaryocytes and platelets the plasmalemma and cytoplasm were well stained with FLA. The cytoplasm of megakaryocytes and the plasmalemma of platelets stained particularly well with FL-RCA. The cytoplasm of both platelets and megakaryocytes showed up strongly with FL-Con A. In the erythroblastic series all three compounds stained the plasmalemma. The remaining cellular components were weakly stained, except the chromatin; that of the late erythroblasts showed up particularly well with FLA. Lymphocytes, monocytes and reticulin cells of the bone marrow were also stained with all three reagents. Reticulin fibres were stained strongly with FLA and FL-Con-A.

Aprotinin↗

PubMedDate not supplied

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides.

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol (PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidum, the microbe that causes syphilis.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators.

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.

Animals↗

PubMed1989

In vivo and in vitro staining of acidophilic neurons as indicative of cell death following kainic acid-induced lesions in rat brain.

An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6h after kainic acid injection. This time was approximately 2-3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.

Animals↗

PubMed2002

Differential fluorescent staining method for detection of bacteria in blood cultures, cerebrospinal fluid and other clinical specimens.

The aim of this study was to evaluate a differential staining method to distinguish gram-positive from gram-negative bacteria in fluorescence. The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein. With this method, gram-positive bacteria appear yellow and gram-negative bacteria appear green. In view of the importance of a rapid aetiological diagnosis in cases of septicaemia, the differential staining method in fluorescence was compared with Gram stain for the detection of bacteria in blood. Of 5,820 blood cultures entered into the study and identified by the Bactec 9120 fluorescent series instrument (Becton Dickinson Europe, France), 774 were positive. Of the 774 positive cultures, 689 yielded only a single organism. The differential staining method in fluorescence detected 626 of the 689 cultures, while Gram stain detected 468. On the basis of these results, the sensitivity of the differential staining method in fluorescence was 90.9%, while that of Gram stain was 67.9%. The difference between the two methods was statistically significant ( P<0.001). The differential fluorescent staining method was more sensitive than Gram stain in the detection of bacteria in blood cultures during the incubation period. This technique provides a rapid, simple and highly sensitive staining method that can be used in conjunction with subculture methods. Whereas subculture requires an incubation period of 18-24 h, the fluorescent staining technique can detect bacteria on the same day that smears are prepared and examined. The differential fluorescent staining method was also evaluated for its ability to detect microorganisms in cerebrospinal fluid and other clinical specimens. The microorganisms were easily detected, even when bacterial counts in the specimens were low.

Bacteria↗

PubMed1985

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains.

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.

Animals↗

PubMedDate not supplied

Developmental changes in the pattern of NADPH-diaphorase staining in the cat's lateral geniculate nucleus.

We examined the pattern of NADPH-diaphorase (NADPH-d) staining in the lateral geniculate nucleus (LGN) of dorsal thalamus in fetal and newborn kittens, and adult cats. This staining visualizes the synthesizing enzyme of nitric oxide (NO), a neuromodulator associated with central nervous system (CNS) development and synaptic plasticity. In the adult, very few LGN cells stained for NADPH-d, and these were restricted to interlaminar zones and ventral C layers. NADPH-d labeled a dense network of fibers and axon terminals throughout the LGN and adjacent thalamic nuclei. The source of such labelling has been reported to be cholinergic neurons from the parabrachial region of the brain stem (Bickford et al., 1993). A very different pattern of staining was observed in prenatal and early postnatal kittens. Between embryonic (E) day 46-57, lightly stained cells appeared throughout the LGN. From this age, through about the first month of life, the number of stained cells in the LGN rose rapidly. The density (cells/mm2) of labeled cells peaked at postnatal day (P) 28 (P28), and was about 150 times greater than the level measured in the adult LGN. After P28, cell staining declined rapidly, and fell to adult levels at P41. The reduction in cell staining that occurred between P35-41 was accompanied by the appearance of fine-caliber fiber staining, similar to that observed in the adult LGN. NADPH-d staining, which reveals the presence of nitric oxide synthase (NOS), and thus NO activity, may reflect two processes. In the adult LGN, the labeling of cholinergic axons arising from the brain-stem parabrachial region coupled with a paucity of the LGN cellular staining suggests that NO operates in an orthograde manner, being co-released with ACh to influence the gain and efficacy of retinogeniculate transmission. By contrast, in developing kitten, NADPH-d staining of LGN cells suggests that NO acts in a retrograde fashion, perhaps playing a role in maintaining associative processes underlying activity-dependent refinement of retinogeniculate connections.

Aging↗

PubMed2001

Intense p53 staining is a valuable prognostic indicator for poor prognosis in medulloblastoma/central nervous system primitive neuroectodermal tumors.

UNLABELLED: Intense p53 immunostaining may predict for a poor prognosis in central nervous system primitive neuroectodermal tumor of childhood. BACKGROUND: Medulloblastoma is a common childhood primary brain tumor. Potential prognostic indicators for patients with local disease are age, extent of resection, and gender. However, none of these are well established. Immunohistologic staining is a potentially useful means to identify high-risk patients. The purpose of this clinical pathologic study was to investigate the prognostic significance of GFAP, synaptophysin, Ki-67, and p53 immunostaining in medulloblastoma/central nervous system primitive neuroectodermal tumors (CNS PNETs.) MATERIALS AND METHODS: The records of 40 patients with CNS PNETs were reviewed. Their surgical specimens were immunostained for p53, glial fibrillary acidic protein (GFAP), synaptophysin, and Ki-67. The p53 specimens were scored blindly for the intensity of staining of nuclei (intense vs weak) and the quantity of cells stained. The Ki-67, GFAP, and synaptophysin specimens were analyzed for quantity of cells stained. RESULTS: Ten patients' specimens stained intensely for the p53 protein. Eleven had weakly staining nuclei. Nineteen specimens had no staining. The patients with specimens that stained intensely had a statistically significant decreased disease free survival (P = 0.03). Mere presence or quantity of p53 nuclear staining did not correlate with disease free survival. Immunohistochemical staining for Ki-67, GFAP, and synaptophysin did not correlate with disease free survival. Clinical parameters of age, gender, and extent of resection also did not approach statistical significance for disease free survival. CONCLUSION: Intense nuclear staining for p53 was the only variable in this clinical pathologic study that reached statistical significance for disease free survival. This suggests that intense staining for p53 may be the most important prognostic indicator for non-metastatic CNS PNETs. p53 Immunostaining with antibodies against p53 in CNS PNETs should be studied in a multi-institutional setting with larger numbers of patients.

Adolescent↗

PubMed1984

Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.

A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr X 10(-3) of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes.

Electrophoresis, Polyacrylamide Gel↗

PubMed2004

Silver stains demonstrating neuroendocrine cells.

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the "older" silver stains and two of the "newer" ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.

Colon↗

PubMed1995

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4-64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.

Biological Transport↗

PubMed1992

Monoclonal antibody to Pneumocystis carinii. Comparison with silver stain in bronchial lavage specimens.

Monoclonal 3F6 anti-Pneumocystis carinii antibody (MAB-3F6) was used to stain cell blocks from 164 bronchial lavage specimens from patients with the acquired immune deficiency syndrome (AIDS) and AIDS-related complex and compared with slides stained with Grocott's modification of the Gomori methenamine silver stain. Pneumocystis organisms were present in 83 of 164 cases using MAB-3F6 stain, whereas Grocott's modified silver stain demonstrated Pneumocystis organisms in 48. MAB-3F6 demonstrated Pneumocystis organisms in 38 cases with negative silver stains, whereas silver stain identified Pneumocystis organisms in only three MAB-3F6-negative cases. Of 70 patients with clinical Pneumocystis pneumonia at the time of the specimen was obtained, 59 had MAB-3F6-positive specimens, whereas 39 had organisms detected using Grocott's modified silver stain. Of 37 patients without clinically apparent Pneumocystis pneumonia any time in their course, 4 had abundant organisms and 33 had negative stains with MAB-3F6. MAB-3F6 detected Pneumocystis organisms in 22 of 31 cases of Pneumocystis pneumonia that had no organisms identified using Grocott's silver stain (X2 = 5.76, P = 0.016). MAB-3F6 immunochemical staining is a more sensitive method than Grocott's modified silver stain to detect Pneumocystis organisms.

Antibodies, Monoclonal↗

PubMed2006

The use of routine special stains for upper gastrointestinal biopsies.

Helicobacter pylori and intestinal metaplasia (IM) are readily seen in hematoxylin and eosin-stained slides of gastric and/or esophageal biopsies, yet many pathology laboratories perform routine special stains on all of these biopsies. We wished to determine if special stains are necessary for every single gastric and/or esophageal biopsy. We prospectively studied 613 gastric and/or esophageal biopsies from 494 consecutive patients. The slides were stained with hematoxylin and eosin, toluidine blue (TB) for H. pylori, and Alcian blue (AB) for IM. The hematoxylin and eosin slide was classed as positive or negative for H. pylori and IM. Then it was determined if the case needed a TB or AB stain. A total of 436 cases (71.1%) were identified as H. pylori-negative and not needing a TB stain, and none was TB+. A total of 126 (20.6%) of hematoxylin and eosin slides were inconclusive for H. pylori and were regarded as needing a TB stain. Twenty of these (15.9%) were TB+. Fifty-one biopsies (8.3%) were regarded as H. pylori+ on hematoxylin and eosin; the TB stain was also positive in 49. IM was present in 113 (18.4%) hematoxylin and eosin biopsies. Hematoxylin and eosin slides were IM-negative in 498 cases (81.2%). The AB stain revealed rare goblet cells in 3 of 498 cases (0.6%). Only one of those biopsies was esophageal, and that had one goblet cell that was missed on hematoxylin and eosin. Only 2 (0.3%) were regarded as needing an AB stain. We conclude that routine special stains for all gastric and/or esophageal biopsies are not required, and hematoxylin and eosin assessment combined with selective ordering of these stains will identify virtually all cases of H. pylori gastritis and intestinal metaplasia.

Alcian Blue↗