A bright field/fluorescent stain for aluminum: its specificity, validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

A bright field/fluorescent stain for aluminum: its specificity,validation, and staining characteristics

A sensitive bright field/fluorescent histochemical staining method has been developed that reveals endogenous aluminum in subcellular structures. The method, achievable within 30 min, is based on phloxine B and phosphotungstic acid, with ethanol differentiation. Hematoxylin is used for nuclear and fast green FCF for cytoplasmic counterstaining. To test the method's specificity, we incubated living neuroblastoma cells overnight in culture media containing aluminum, calcium, iron, copper or zinc, or no added metal ions. After fixing the cells and applying the staining method, only cultures exposed to aluminum stained magenta. Applying the method to paraffin embedded tissue sections pretreated with one of two chelating agents that remove aluminum demonstrated less magenta staining in the chelated sections than in adjacent unchelated sections. Immersing sections overnight in solutions containing exogenous aluminum had no observable effect on staining for endogenous aluminum; therefore, it is unlikely that any exogenous aluminum present in histological reagents would alter the method's staining results.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes non-specific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidin-biotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

Summary The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes nonspecific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidinbiotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

Ultracytochemistry of calmodulin binding sites in myocardial cells by staining of frozen thin sections with colloidal gold-labeled calmodulin

Calmodulin (CaM) has been implicated as a multifunctional regulator of Ca2+ in the cytoplasm of cells. We have recently introduced biologically active colloidal gold-labeled CaM as a marker for identifying potential CaM binding sites (unoccupied by endogenous CaM at the time of fixation) by electron microscopy and have stained frozen thin sections of rat cardiac muscle with this conjugate. In the presence of Ca2+, gold particles indicating CaM binding sites were found localized on the sarcoplasmic reticulum, mitochondria, and gap junctions. Control tissue sections treated with EGTA or exposed to excess amounts of unlabeled native CaM before staining showed no binding. We believe that cytochemistry of potential CaM binding sites revealed by staining with labeled exogenous CaM is useful in correlating known biochemical reactions of CaM with particular cell activities.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

Histochemical staining of exogenous chromium and iron with the catalytic oxidation of phenylamines

A highly sensitive and specific method for staining exogenous chromium and iron in tissues is described. This method is superior to conventional complex-forming methods with regard to its sensitivity and specificity for these metals. The staining reaction is based on the metal-catalysed oxidation of phenylamines. Tissue sections were incubated in a medium containing hydrogen peroxide and phenylamines, p-phenylenediamine or phenylhydrazine. Results obtained from test-tube experiments concerning the catalytic activities of metals indicated that the staining reactions depends on the activities of metals in tissues.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A simplified aluminum stain in paraffin sections of bone from hemodialysis patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

Histochemical staining of exogenous chromium and iron with the catalytic oxidation of phenylamines

Summary A highly sensitive and specific method for staining exogenous chromium and iron in tissues is described. This method is superior to conventional complex-forming methods with regard to its sensitivity and specificity for these metals. The staining reaction is based on the metalcatalysed oxidation of phenylamines. Tissue sections were incubated in a medium containing hydrogen peroxide and phenylamines, p-phenylenediamine or phenylhydrazine. Results obtained from test-tube experiments concerning the catalytic activities of metals indicated that the staining reactions depends on the activities of metals in tissues.     

A Modification of the Fite Formaldehyde (Fite I) Method for Staining Acid-Fast Bacilli in Paraffin Sections

For maximal demonstration of acid-fast bacilli in tissue sections it is necessary to avoid sequences of reagents which, first, affect the integrity of the complex upon which acid-fastness depends, and, second, extracts it from those bacillary elements which have been made vulnerable by age or other factors. The greatest damage occurs during dewaxing of paraffin sections by xylene and alcohols; the older and more decrepit bacilli being especially affected. In the technic presented, which is a modified combination of two processes devised by Fite, sections are deparaffinized by a protective mixture of rectified turpentine and heavy liquid petrolatum (2:1) and blotted to water. Staining is with Fite's new fuchsin (magenta III) solution, overnight at room temperature. The sections are then treated with reagent grade formaldehyde, which turns the color of the bacilli deep blue-black, followed by an aqueous sulfuric acid decolorizer, the potassium permanganate-oxalic acid sequence, and a modified Van Gieson counterstain, nuclear staining with hematoxylin being omitted. For total demonstration of all stainable bacilli, restorative treatment in the turpentine-oil mixture before staining is sometimes required, most frequently with leprosy material but also with some tuberculosis lesions.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Isolation of Avian Trypanosomes by Selective Centrifugation and Subsequent Processing for Electron Microscopy

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10–20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

A re-evaluation of cytoplasmic gelsolin localization

Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large-scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co-localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different levels of reactive endogenous cytochrome c in mitochondria rich cells revealed by diaminobenzidine staining

Light microscopic examination of rat and mouse tissues incubated in a medium containing 3,3'-diaminobenzidine (DAB) and catalase revealed that cells known to possess abundant mitochondria (hepatocytes, cardiomyocytes, renal proximal and distal tubular cells, parietal cells of gastric mucosa, and retinal photoreceptor cells) were stained with different intensity: from moderate (parietal cells, cardiomyocytes, renal distal tubular cells) to weak (hepatocytes, renal proximal tubular cells) or even negative (photoreceptors). When exogenous cytochrome c was added to the incubation medium, all these cells displayed quite uniform, strong staining, indicating a comparable activity of cytochrome oxidase. Since DAB is oxidized directly by cytochrome c which in turn undergoes reoxidation by cytochrome oxidase, the observed differences of staining intensity in the absence of exogenous cytochrome c are postulated to result from different content of reactive endogenous cytochrome c in mitochondria of the investigated cells.     

Immunohistochemical detection of mammary tumor virus antigens in sweat and sebaceous glands of mice

Mammary tumor virus (MTV) antigens in both sexes of GRS/A, SHN and C3H mice were examined in the sweat and sebaceous glands by immunoperoxidase technique using antiserum against gp52, envelope protein, or p27, core protein. Balb/c mice were used for reciprocal foster nursing with these inbreds to discriminate the expression of endogenous MTV from that of exogenous MTV. Both antigens were first detected around the age of 4 months in the sweat glands of mice with endogenous GR- or SHN- MTV. A linear staining of gp52 was seen along the luminal borders of glandular cells, and the reaction products for gp52 were demonstrated on the apical cell membranes, where no virion could be seen ultrastructurally. A diffuse staining of p27 was found in the cytoplasm of some glandular cells, where MTV particles could not be detected. In the sebaceous gland of the same mice, however, only p27 was first detected at the age of 60 days. A dot-like staining of p27 was found in the perinuclear region of some glandular cells, where an aggregation of intracytoplasmic A particles could be seen under an electron microscope. These positive stainings were unrelated to sex. In such skin appendages of all examined C3H mice and Balb/c mice with GR- or SHN- MTV, no antigen expression could be seen up to the age of 500 days. Therefore, some genes might be able to regulate the expression of endogenous MTV antigens in the skin appendages, while their glandular cells would have no receptor for exogenous MTV, namely the so-called "milk factor".     

Histochemical demonstration of aluminum and iron deposition in pulmonary bony tissues in three cases of diffuse pulmonary ossification

Diffuse pulmonary ossification is a rare condition. We examined three cases of it in Japan, and attempted histochemically to stain for deposition of aluminum and iron in bony tissues. The patients were all female, and in their mid-twenties, mid- eighties, and later teen years. One of the patients had been exposed to heavy metals in her work involving heavy-metal analyses for 18 months. Aluminum staining and Berlin blue staining for iron were performed with dewaxed, undecalcified sections of pulmonary tissues from these three cases. Interestingly, all pulmonary bony tissues from the three cases examined exhibited linear regions of both aluminum and iron deposition in the calcifying fronts or the cement lines of bones. The patient exposed to heavy metals exhibited the most severe aluminum and iron deposition, and also exhibited positive reaction for both aluminum and iron in elastic fibers of blood vessels. Foreign body granulomas with multinucleated giant cells exhibiting elastophagia were also found in this case. This phenomenon, "endogenous pneumoconiosis", appeared to have been the cause of pulmonary hemorrhage in this case, resulting in focal heavy hemosiderosis. It is of great interest that identical patterns of aluminum and iron deposition in hemodialysis patients were found in these three cases, This is the first report on histochemical demonstration of aluminum and iron deposition in diffuse pulmonary ossification, and detailed analysis of additional cases is needed.     

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.     

Staining Tomato Fruit Cuticle and Exocarp Tissues

Immature fruit of tomato, Lycopersicon esculentum (Celebrity), was examined to observe the cuticle, its interface with the epidermis, and the general histology of the outer exocarp. Paraffin sections were stained first with Bismarck brown Y. Structures already stained in various hues of brown were stained again with either azure B, aluminum hematoxylin and alcian blue 8GX, or the periodic acid-Schiff (PAS) reaction. Bismarck brown-azure B displayed the cuticle in strong contrast with subjacent tissue; however, nuclei were not easily identified at low magnification. Bismarck brown-hematoxylinalcian blue produced a sharply contrasted combination of yellow cuticle, bright blue cell walls and purple nuclei. Nuclei stained purple with hematoxylin were easily identified at × 100. Bismarck brown-PAS stained the cuticle golden brown and subjacent tissues magenta red. Surprisingly, epidermal cells stained specifically and intensely with PAS while pretreatment with an aldehyde blockade and omission of periodic acid prevented staining of all other tissues.     

A Simple Method of Staining Juxtaglomerular Granules in the Rat Kidney for High Resolution Light Microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 μm) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicro-tome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 × or a 100 × objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.