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Immunoperoxidase staining of fine needle aspiration specimens previously stained by the Papanicolaou technique.

The use of immunoperoxidase techniques was investigated in 21 fine needle aspiration (FNA) cytology smears that had been previously stained by the Papanicolaou technique. The retrospectively selected slides were destained before applying the immunostain, utilizing antisera to calcitonin, prostatic acid phosphatase (PrAP), prostate-specific antigen (PSA), alpha-lactalbumin (AL), S-100 protein (S-100), carcinoembryonic antigen (CEA), common leukocyte antigen (LA), epithelial membrane antigen (EMA) and alpha-fetoprotein (AFP). Positive results were obtained with six of nine small-cell carcinomas of the lung stained with EMA, all three colonic carcinomas stained with CEA, one of two prostatic carcinomas stained with PSA and PrAP, one of two lymphomas stained with LA and the one medullary thyroid carcinoma stained with calcitonin. Negative staining results were observed in the one melanoma stained with S-100, the two breast carcinomas stained with AL and the one hepatocellular carcinoma stained with AFP. These results indicate that immunostaining can be a helpful diagnostic tool in diagnosing some fine needle aspirates using smears previously stained with the Papanicolaou stain.

Biopsy, Needle↗

Staining of the Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye "Stains-all".

The Ca2+-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, have all been shown to stain dark blue or purple with the cationic carbocyanine dye "Stains-all", while most proteins stain red or pink. Stains-all staining of these Ca2+-binding proteins was 50-100% more intense than Coomassie blue staining, and the blue stained calsequestrin and troponin C could be detected in muscle extracts. Densitometric scans of Stains-all-stained gels revealed that interaction of the dye with Ca2+-binding proteins changed the absorption spectrum of the dye. The dye-protein complex absorbed maximally at 615 nm, reflecting a state that results from the binding of individual dye molecules at anionic sites. Stains-all also interacted with undenatured Ca2+-binding proteins in aqueous solution forming a complex absorbing maximally at 600 nm. A simple assay for Ca2+-binding proteins has been developed using this property. These results suggest that the interaction of the dye with anionic sites within these Ca2+-binding proteins produces the dye-protein complex which absorbs at 600-615 nm. Stains-all staining of these Ca2+-binding proteins will be useful in their identification and purification from various tissues. It might also be a valuable tool in the identification of potential Ca2+-binding proteins.

Animals↗

The TP-levanol fast cyanine 5RN staining procedure for proteins of the myosin-fibrin group. Fibrin staining and other remarks.

The tannic acid-phosphomolybdic acid-Levanol (Supranol) Fast Cyanine 5RN (TP-L) procedure for staining muscle cells and blood platelets was used because, with this method, proteins of the myosin-fibrin group should be selectively stained. However, in human blood and blood plasma clots and in vivo thrombi, fibrin was not stained. Blood platelets probably due to their content of contractile proteins were very well stained. Apparent fibrin staining in human autopsy thrombi may be due to the staining of disintegrated platelets and the absorbance of fibrin by stained hemoglobin. Problems encountered using Nuclear Fast Red as the nuclear stain were solved by changing the dye concentration or by using a differentiating agent. Myosin staining by the TP-L method depended on the pH of the tannic-acid solution used. Raising the pH to 7.4-8.0 changed the staining result, and collagen fibers were then stained.

Animals↗

The mechanism accounted for the silver staining "fast technique" development and its histochemical meaning: I. The correlation between the silver staining intensity and the tissue bound ferric ion and some other tissue histochemical reactivities.

The mechanism accounted to accomplish the silver staining "fast technique" on tissues sections was studied towards the correlation among histophotometric measures concerning the silver staining intensity and the intensity provided by some histochemical reactions performed on spleen and liver sections from rats and pigs. By treating previously these histological sections with thioglycolate or oxalate solutions in progressive concentrations and afterwards subjecting them to a silver staining "fast technique", it was demonstrated that the silver staining intensity decreases proportionally to the thioglycolate or the oxalate solution concentration. The regression line of the silver staining intensity on the thioglycolate or the oxalate solution concentration was established, as well as its regression coefficient. On the other hand, on histological sections previously subjected to the thioglycolate or the oxalate treatment, some histochemical techniques were performed and the histophotometric measures concerning the intensity of each histochemical technique used were taken and its regression line, as well as its regression coefficient were established. By comparing the silver staining intensity regression line (or its regression coefficient) with the regression lines established for the histochemical techniques used, it was tested the correlation between the silver staining and the reactivity of some reactive groups contained into the tissues. In this manner, the influence of such reactive groups on the silver staining development was tested. The results show that there is no correlation between the silver staining intensity and the reactivity degree of the reducing groups (-SH- and carbonyl group), the 1-2-glycolic group, as well as of some protein reactive groups (phenol, imidazole, carboxylic groups). On the other hand, taking into account the respective regression lines and its regression coefficient, the comparison between the silver staining intensity and the Prussian blue reaction intensity shows a close relationship between them. This finding suggests that for the silver staining "fast technique" the tissue bound ferric ion accomplishes a very important role and can be accounted for its mechanism and its histochemical meaning.

Animals↗

Comparison of Papanicolaou's stain with the Gomori methenamine silver (GMS) stain for the cytodiagnosis of Pneumocystis carinii in bronchoalveolar lavage (BAL) fluid.

The cytodiagnosis of Pneumocystis carinii (PC) in bronchoalveolar lavage (BAL) fluids has traditionally required a special stain such as Gomori's methenamine silver (GMS) stain. Recent reports indicate that identification of foamy alveolar casts (FACs) with Papanicolaou's (Pap) stain may provide a sensitive and less complicated way of making the diagnosis. To confirm these observations, results on a series of 318 BALs were reviewed. PC was identified on 65 (20%) specimens from 54 patients. Pap stains and GMS stains were positive on 56 (86%) of these BALs. Pap stains were positive on seven (11%) specimens that had negative GMS stains. PC was later confirmed on these specimens by other methods. Only two (3%) BALs had positive GMS stains and negative Pap stains. The results of this study confirm other reports that show that PC can be sensitively diagnosed with the Pap stain. The authors suggest that routine special stains for PC are unnecessary on BALs.

Bronchoalveolar Lavage Fluid↗

Vital staining properties of neutral red. Vital staining of cornea and conjunctiva.

Vital staining by instillation in the conjunctival sac of 1% neutral red in 54 normal and 153 differently affected eyes was studied by slit lamp examination. Of these, 134 eyes were after-stained by a mixture of 1% tetrazolium and 1/4% alcian blue and 73 eyes were after-stained by a mixture of 1% rose bengal and 1% fluorescein. Microscopy of a further 10 conjunctival scrapings and 40 mucous conjunctival threads disclosed neutral-red-stained inclusion bodies in the cytoplasm of the epithelial cells and the granulocytes. In the slit lamp the cornea and the conjunctiva were seen to be stained only rarely and, if so, poorly by neutral red, both in normal and affected eyes. Better staining was obtained with tetrazolium, and the best with rose bengal. The plica semilunaris, Marx' line, and the mucous thread were stained by neutral red, more intensely by rose bengal, and the least by tetrazolium. The inferior fornix and the tarsus were rarely stained by any of the three dyes. Neutral red seems to offer no diagnostic advantages over the vital stains with which the dye has been compared in the present study. More particularly we found no pathological processes that were stained more intensely by neutral red than by the other vital stains.

Cataract↗

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.

Cytological Techniques↗

Analytical electron microscopy of chlorhexidine-induced tooth stain in humans: direct evidence for metal-induced stain.

Using analytical electron microscopy, we directly tested the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. Plaque scraped from the teeth of water-treated individuals or chlorhexidine-treated "non-stainers" was non- or lightly-stained and contained low concentrations of sulfur and transition metals. Tooth scrapings from heavily-staining chlorhexidine-treated individuals consisted of distinct unstained and stained regions. The stained regions were organic but were in close proximity to mineralized areas. Enhanced levels of sulfur and transition metals, particularly iron, were found in stained regions, whereas unstained regions contained low sulfur and metal levels similar to the water-treated or non-staining individuals. Excluding decreased mineralization, the major elemental change in heavily-stained plaque was an increase in sulfur, and to a lesser extent, iron and other transition metals. Sulphur and iron levels were directly correlated. Following chlorhexidine treatment with iron supplementation, staining was enhanced, the Fe/S ratio increased, and sulfur and iron remained correlated. These data support the hypothesis that transition metals and sulfur are the cause of chlorhexidine-induced tooth stain. The data are consistent with chlorhexidine treatment altering the incorporation into plaque of a natural sulfur-containing organic component of saliva or bacteria. This natural component appears to readily interact with transition metals, particularly iron, producing stained material.

Chlorhexidine↗

A comparison of the use of Papanicolaou-stained cervical cytological smears with Gram-stained vaginal smears for the diagnosis of bacterial vaginosis in early pregnancy.

Our objective is to compare the efficacy of using Papanicolaou (PAP)-stained cervical cytology smears with a standardized method of interpreting Gram-stained vaginal smears for the diagnosis of bacterial vaginosis (BV) in pregnancy. High vaginal smears were Gram-stained and examined by a single observer to characterize 3 grades of vaginal flora and diagnose BV. Cervical smears were PAP-stained and examined for characteristic patterns of vaginal flora including evidence of BV by either a number of cytotechnicians or a single cytopathologist. The results of the 2 methods were compared. Seven hundred and forty-seven women attending an antenatal clinic in a district general hospital who consented to have a smear of vaginal secretions and cervical cytology in early pregnancy. The main outcome measure is the diagnosis of BV by different methods in a pregnant population. Compared with the Gram-stain method for the diagnosis of BV, there was good agreement between PAP-stain interpretation by a single observer but the agreement was not as good with PAP-stain interpretation by multiple cytotechnicians. When the grades were consolidated to normal (grade I) and abnormal flora (grades II and III), compared to Gram-stained smears, PAP cytology undertaken by several cytotechnicians had a sensitivity of 80.7% and a specificity of 90.7%. The sensitivity and specificity increased to 87% and 97%, respectively, when the PAP-stained smears were read by a single cytopathologist. Using kappa scores, only those readings made by a single cytopathologist were reliable. The setting in a cytopathology laboratory comprises multiple cytotechnicians, so that PAP-stain analysis of vaginal smears for the diagnosis of BV is likely to provide results which are less reliable than those obtained by Gram staining. The latter should be the first choice and every effort should be made to set up this service.

Cervix Uteri↗

Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique.

We have recently shown that a few nanograms of protein separated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels can be detected by reverse-staining, exploiting the precipitation reaction between zinc(II) and imidazole. Modifications of this method have also been generated to detect gel-isolated nucleic acids and bacterial glycolipids. Because there is no recourse to chemical modifiers, the reverse-staining technique has been valuable when micropreparing these biomacromolecules for later use or characterization. The mechanism underlying the reverse-staining effect, however, remains incompletely understood and this has prevented a further generalization of the technique. Here, we have conducted physicochemical experiments and identified zinc imidazolate (ZnIm2) as the main component of the precipitate that forms along the surface of zinc-imidazole reverse-stained gels. Many staining effects observed when gels containing electrophoretically separated biopolymers are subjected to zinc-imidazole stains have been rationalized. The reverse-staining method has been vastly generalized, now allowing the detection of proteins and glycolipids as well as complexes of these macromolecules in native gels. We demonstrate the application of the reverse-staining technique in situations where Coomassie blue or silver staining was inappropriate or failed to produce detection of the species of interest. The present generalization of the reverse-staining method facilitated the characterization of biomacromolecular interaction partners in mixtures of bacterial glycolipids and human tears.

Bacteria↗

Comparison of a fluoresceinated lectin stain with triple staining for evaluating acrosome reactions of dog sperm.

In this study a technique which utilized a fluoresceinated lectin and a fluorescent supravital stain was compared with the conventional triple stain technique for evaluating the viability and acrosomal status of dog sperm. Ten semen samples obtained from 6 normal beagle dogs were evaluated after incubation in vitro with canine capacitation medium. Sperm viability and acrosomal status were assessed at 0, 4, and 7 hours of incubation. Both staining techniques were capable of detecting the increase in spontaneous acrosome reactions which occurs during in vitro capacitation of dog sperm. High positive correlations were observed between the fluorescent stain and the triple stain in the mean values for the percentage of viable sperm and for the percentage of acrosome-reacted sperm among the viable sperm (r = 0.91, r = 0.97, respectively). However, the fluorescent staining techniques could be carried out much more rapidly than the triple stain technique, and the slides prepared with fluorescent stain were more easily scored because of the higher intensity and greater consistency of staining. These characteristics make the fluoresceinated lectin and the fluorescent supravital stain superior for evaluating acrosome reactions and viability of dog sperm.

Acrosome↗

SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein.

We have developed two new fluorescent dyes, SYPRO Orange protein gel stain and SYPRO Red protein gel stain, to detect proteins in electrophoretic gels. Stained protein bands can be excited by ultraviolet light at approximately 300 nm, or at visible wavelengths, with excitation maxima of 472 nm for the Orange stain and 547 nm for the Red stain. Detection can be documented with sensitivity similar to that achieved with silver stain, using standard UV transilluminators and Polaroid 667 black and white film, CCD cameras, or commercially available laser scanners. Staining with these dyes is noncovalent and is accomplished using a one-step procedure. Protein gels do not require fixation steps prior to incubation with the dyes. Staining is complete 30 to 60 min following electrophoresis, with no destaining required. Staining can also be accomplished by including dye in the running buffer; in this case a brief one-step destaining procedure follows electrophoresis. The dyes appear to bind to the detergent coat surrounding proteins in sodium dodecyl sulfate (SDS) denaturing gels; thus, staining in such gels is not strongly selective for particular polypeptides. Fluorescent signals are relatively photostable, allowing multiple photographs of gels to be taken without significant signal reduction.

Animals↗

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.

Cell Line↗

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention.

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.

Chymotrypsin↗

Pathologic evaluation of inguinal sentinel lymph nodes in vulvar cancer patients: a comparison of immunohistochemical staining versus ultrastaging with hematoxylin and eosin staining.

OBJECTIVES: To evaluate the value of immunohistochemical (IHC) staining of inguinal sentinel lymph nodes (SLN) found to be negative for metastatic disease by ultrastaging with hematoxylin and eosin (H&E) staining. METHODS: An IRB approved study identified 29 patients who had undergone an inguinal sentinel lymph node dissection for squamous cell carcinoma of the vulva. All sentinel lymph nodes found to be negative for metastatic disease based on ultrastaging with H&E staining were reevaluated with pancytokeratin antibody (AE1/AE3) immunohistochemical (IHC) staining to detect micrometastasis. RESULTS: Twenty-nine patients with squamous cell carcinoma of the vulva underwent an inguinal sentinel node dissection. Nineteen patients had inguinal dissections negative for metastatic disease, 2 patients had bilateral inguinal metastasis, and 8 patients had unilateral inguinal metastasis. A total of 42 groin dissections with SLN biopsies were performed; 12 groins were positive for metastatic disease and 30 were negative based on ultrastaging with eosin and hematoxylin staining. A total of 107 sentinel lymph nodes (2.5 SLN per groin) were obtained, of which 18 SLN contained metastatic disease identified by ultrastaging and staining with H&E. Two SLN contained micrometastasis less than 0.3mm in size and 16 SLN contained metastasis greater than 2mm in size. Eighty-nine SLN found to be negative for metastasis by ultrastaging with H&E staining were also negative for micrometastasis on evaluation with pancytokeratin antibody AE1/AE3 IHC staining. CONCLUSIONS: The addition of immunohistochemical staining to ultrastaging with H&E staining in the pathologic evaluation of inguinal sentinel lymph nodes does not increase the detection of micrometastasis in patients with primary squamous cell carcinoma of the vulva.

Carcinoma, Squamous Cell↗

Papanicolaou stain: Is it economical to switch to rapid, economical, acetic acid, papanicolaou stain?

OBJECTIVE: To standardize an inexpensive and rapid Papanicolaou staining technique with limited ethanol usage. STUDY DESIGN: Smears from 200 patients were collected (2 per patient) and fixed in methanol. Half were subjected to conventional Papanicolaou and half to stain ing with rapid, economical, acetic acid Papanicolaou (REAP) stain. In REAP, pre-OG6 and post-OG6 and post-EA36 ethanol baths were replaced by 1% acetic acid and Scott's tap water with tap water. Hematoxylin was preheated to 60 degrees C. Final dehydration was with methanol. REAP smears were compared with Papanicolaou smears for optimal cytoplasmic and nuclear staining, stain preservation, cost and turnaround time. RESULTS: With the REAP method, cytoplasmic and nuclear staining was optimal in 181 and 192 cases, respectively. The staining time was considerably reduced, to 3 minutes, and the cost per smear was reduced to one fourth. The staining quality remained good in all the smears for > 2 years. CONCLUSION: REAP is a rapid, cost-effective alternative to Papanicolaou stain. Though low stain penetration in large cell clusters is a limitation, final interpretation was not compromised.

Acetic Acid↗

Comparison of acridine orange stain with culture and gram stain of needle aspirate in experimental Pseudomonas pneumonia.

The sensitivity and specificity of culture, acridine orange stain, and Gram stain were determined using needle aspiration (NA) material obtained from 82 rats with acute Pseudomonas aeruginosa pneumonia and 18 control rats. Lungs were then processed for either bacterial quantitation or histopathologic examination. NA culture proved to be the most sensitive and specific (55 and 100%, respectively). Sensitivity of acridine orange stain was 40%, whereas Gram stain was only 29%. The specificity of each stain was at least 94%. Lung bacterial concentrations influenced the sensitivities of all three techniques, with better sensitivity found in NA samples obtained from lung with bacterial concentration of at least 10(4) colony-forming units (cfu) of P. aeruginosa. Acridine orange and Gram stain results were similar except in NA samples from lung with bacterial concentration of less than 10(4) cfu in which acridine orange stain was more sensitive. The presence of stains identifying bacteria collected from animals with sterile NA culture was found in a small but significant number of samples, suggesting the presence of nonviable though stainable organisms. Use of all three techniques (culture, acridine orange stain, and Gram stain) increased sensitivity to approximately 70% with minimal decrease of specificity.

Acridine Orange↗

Comparative analysis of an improved thioflavin-s stain, Gallyas silver stain, and immunohistochemistry for neurofibrillary tangle demonstration on the same sections.

An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.

Aged↗