Staining Steps Research Articles

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the SOS monosomes and the large (608) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the “native” eukaryotic ribosome 3-D model.

Batch Staining for Demonstration of Gram Positive and Gram Negative Bacteria in Tissue Sections

Abstract A cost effective, reliable method for the demonstration of gram positive and gram negative bacteria in tissue sections is described. This procedure, a modification of the Brown-Hopps method, utilizes a slide basket and staining dishes for all steps of staining and differentiation to eliminate time consuming handling of individual slides. Stains and reagents can be reused for further savings on costs and preparation time. (The J Histotechnol 15:49, 1992)

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convinient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.

抗酸菌のＧｒａｍ染色性を左右する加温染色操作の効果

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.

Analysis of brain and myelin lipids by high-performance thin layer chromatography and densitometry

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol:acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.

Immunohistochemical analysis of the topographical relationship between the estrogenand progesterone receptor in five human breast cancers

A technique is described which allows precise assessment of the topographical relationship between the estrogen receptor (ER) and the progesterone receptor (PR) in the same histological section. It is based on the analysis of the results of immunohistochemical double staining by computer-assisted image processing. Five human ductal breast cancers were examined. The simultaneous demonstration of both receptors consists in the following principal steps: The primary antisera against the ER (monoclonal rat antibody) and the PR (monoclonal mouse antibody) are incubated simultaneously, but only the anti-ER antibody is demonstrated in the first staining step by using a goat anti-rat antibody as the linking antibody and the PAP complex from the rat, both antisera from the ER-ICA kit. The result is stored as a digitized grey image ("1. object image"). Then the colored end product and the residual peroxidase activity of the PAP complex are removed. In the second staining sequence the anti-PR-antibody is demonstrated by using a rabbit anti-mouse antibody as the linking antibody and the PAP complex from the mouse. The result is exactly positioned and also stored as a digitized grey image ("2. object image"). Though antibodies raised in different species were used, cross-reactivity could not be avoided. Grey values generated by cross-reactivity between the different antibody systems are evaluated in negative controls and are eliminated in the object images. The remaining (specifically stained) structures of both object images are copied into a final image so that the topographical relationship of the ER and the PR becomes obvious. The results show that in the five carcinomas investigated three types of receptor-positive tumour cells can be distinguished: Cells which coexpress the ER and the PR (1), cells which express either the ER (2) or the PR (3). The number of tumour cells showing one of these expression patterns varies from tumour to tumour.

Ultrasensitive polychromatic silver staining of sodium dodecyl sulfate‐polyacrylamide gels with the Gelcode system using the PhastSystem Development Unit

The Gelcode color-based silver staining system, an improved formulation based on the original publication by Sammons et al. (Electrophoresis 1981, 2, 141-147) has been adapted to automated rapid staining in the PhastSystem Development Unit. The use of elevated temperatures in the fixation, washing, staining, and stabilization steps of the protocol reduces the total time of the process from 18 h to 1 h. The limit of detection, which is at least tenfold more sensitive than the silver staining protocol recommended for the PhastSystem, corresponds to 0.05-0.1 ng of protein per band. The method is applicable to both one- and two-dimensional polyacrylamide gels.

A Rapid Screening Technique for the Detection of Spermatozoa

Phase contrast microscopy has been used for some time to search for and identify spermatozoa. An enhancement to the technique using xylene in conjuction with phase contrast microscopy is discussed. The method has been found to save time by allowing identification of spermatozoa with xylene-enhanced phase contrast microscopy in many cases that would have been unsuccessful using the normal "dry" phase contrast microscopy techniques. This eliminates a staining and reexamination step.

Sodium dodecyl sulfate electrophoresis in the first dimension and isoelectric focusing in the second dimension in a single thin‐layer gel, followed by activity stain for glycosyltransferases

AbstractA method has been developed for the two‐dimensional separation of proteins in a single thin‐layer gel, using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the first dimension and isoelectric focusing in the second dimension. After two‐dimensional electrophoresis of concentrated culture supernatants from Streptococcus mutans, the gels were directly stained with Coomassie Brilliant Blue R‐250 for protein and with periodic acid‐Schiff base reagent for glycoprotein. Glycosyl‐transferases were detected by incubating the gels in a sucrose‐containing buffer, either by visual observation of white spots of the product polysaccharides or by periodic acid‐Schiff staining of the products. All the procedures, including the staining steps, were done in a single gellayer covalently bound to a glass plate. The efficient removal of sodium dodecyl sulfate and renaturation of glycosyltransferases in the gel layers rendered this a simplified and rapid method for the relative molecular mass and isoelectric point determinations in the crude preparations.

The Rose Bengal Assay for Monoclonal Antibodies to Cell Surface Antigens: Comparisons with Common Hybridoma Screening Methods

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetric assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.

Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy.

A method is presented whereby the staining of intracellular structures with immunogold probes for electron microscopy can be evaluated at the light microscopic level. Methanol-fixed monolayers of cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were stained for cytokeratins using a two-step immunogold technique consisting of primary anti-keratin antibody followed by gold-labeled secondary antibody. Bound immunogold probe was then visualized with a fluorescent tertiary anti-immunogold probe antibody. Fluorescence microscopy of the whole cell monolayers showed a typical keratin cytoskeleton. The extra staining step did not interfere with subsequent fixation, embedding, and sectioning for electron microscopy, which showed cytoplasmic intermediate filaments decorated with colloidal gold. Using this method, it should be possible to manipulate parameters critical to staining with immunogold probes and to evaluate the labeling without necessitating repeated time-consuming electron microscopic processing. The method also provides a useful correlation between the light microscopic and ultrastructural labeling patterns of immunogold probes.

An improved method for the demonstration of fibrin in biopsies of rabbit skin homografts.

Tissues embedded in resin are convenient for routine use when the presence or absence of fibrin in them is to be confirmed using the electron microscope. To visualize fibrin using the light microscope, sections (1.0-2.0 micron) from such specimens should be stained with Methylene Blue-Azure II-Basic Fuchsin (MBBF). Staining with MBBF is more controllable than with other methods and it requires only two short staining steps. Compared with Giemsa, MBBF provides a polychromatic, as opposed to a monochromatic end-result, sharply contrasting fibrin (blue) against collagen (pink-violet).

Fluorescent monitoring of proteins during sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blotting

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100–200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.

Human brain in tissue cultures. VI. Presence of glial fibrillary acidic protein in subcultivated human fetal brain cells as demonstrated by immunofluorescent and immunoperoxidase staining.

Glial fibrillary acidic protein (GFAP) was present in cell cultures derived from human fetal brain tissue as determined by indirect immunofluorescence (IF) and immunoperoxidase (IP) staining using rabbit anti-human GFAP antisera. The IF and IP techniques were comparable in localizing the cytoplasmic distribution and the frequent perinuclear concentration of GFAP in brain cells. The horseradish peroxidase technique was more sensitive and a 1:20-1:40 dilution of anti-GFAP serum could be applied in the initial step of the peroxidase staining as compared to a 1:10 dilution of anti-GFAP serum for IF staining. Sequential studies of subcultivated human fetal brain cell lines by these techniques indicated that some brain cell lines become GFAP-negative rapidly, whereas other cell lines remain GFAP-positive for no less than ten subcultivations in vitro. GFA protein was never present in any PML-SV40-transformed human brain cells.

Immunoenzymatic methods for the study of surface bound immunoglobulins in mice

Methods are described for the study of lymphocyte surface bound immunoglobulins (Ig) for light microscopy experimentation. a) Live peripheral lymph node cells (LNC) or spleen cells were reacted in suspension under capping conditions with rabbit anti-mouse Ig antibodies. After washing the cells were cytocentrifuged, fixed and relabeled with either peroxidase-(PO) of fluorescein-(Fl) conjugated anti-rabbit IgG antibodies (Method SC). The staining was heterogeneous and three main categories of lymphocytes were distinguished on the basis of a characteristic surface label distribution (1) all label within distinct caps (caps), (2) all label evenly distributed over the cell surface (rings), and (3) an intermediate pattern where only part of the label was concentrated over one pole (polarized). b) LNC or spleen cells were treated for the simultaneous detection of lymphocyte surface Ig and cytoplasmic Ig of plasma cells. For this, live cells were cytocentrifuged, and after fixation were labeled with PO-conjugated antibodies. Lymphocytes stained in a continuous ring and were easily distinguished on morphological grounds from the intense cytoplasmic stain of plasma cells (Method D). Positive lymphocyte counts done by this method compared favorably with those obtained by Method SC. Comparisons of Fl- and PO-conjugated reagents were done by either Method SC or D, by scoring total positive cells. Although either reagent gave closely similar percentage values, the lymphocytes stained by the immunoenzymatic technique contrasted sharply from negative cells and were as a result more easily counted. Other practical advantages of immunoenzymatic staining over immunofluorescence are (1) the simultaneous visualization of cell morphology and type of label distribution over the cell surface, (2) cell scoring can be done rapidly on a large population sample, and (3) stained slides can be reexamined on subsequent days. A common advantage of Method SC for both immunofluorescence and immunoenzymology is that antibody labeled slides can be stored before the final staining step.

Combined immunofluorescent staining for the simultaneous detection of anti-DNP antibody and immunoglobulin class in the same cell

A technic was presented combining direct and indirect immunofluorescent staining (IFS) to identify cells containing anibody to a haptenic group and the immunuoglobulin (Ig) class of that antibody. Fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate were conjugated with IgG prepared from various antisera by precipitation with 1.4 M (NH 4) 2SO 4 and DEAE-cellulose chromatography. The reagent in the first staining step contained rhodamine-conjugated antibodies to a specific Ig class and dinitrophenylated human γ-globulin. After washing, the tissue section was stained with fluorescein-conjugated IgG prepared from an antiserum to dinitrophenylated bovine γ-globulin. Fluorescence microscopy revealed either Ig class or antibody to DNP or both in the same cell. Combined IFS was simpler and required less time to obtain results than other technics previously used to identify antibody and Ig class in a single cell.

Effect of G-banding techniques on the ultrastructure of human chromosomes.

The ultrastructure of human chromosomes is studied after each step of the G-banding technique. Without staining the primary coils are shown. After the first and second G-band staining steps, an apparent destruction of the original structure takes place. Dense loops of fibrils, partly arranged in bundles, in G-band regions can only be seen after the final Giemsa staining. The rest of the chromatid also consists of fibrils, but not so compact. The diameter of the fibrils is 300–600 A. The centromere region consists of parallel fibrils. Identical chromosomes were studied with light and electron microscope, threedimensionally depicted and evaluated by means of the equidensity method.

A simple fixation-decalcification and iron staining technic for bone marrow specimens.

A method for processing bone marrow specimens is described. It telescopes the three steps of fixation, decalcification, and iron staining into a simple one-step method using only one working solution. Cellular detail is excellent The iron stain appears to be superior in sensitivity to conventional staining technics. Iron is easily demonstrated in the mounted, unstained, deparaflinized slide and also in the slide stained with hematoxylin and eosin.

Electrophoresis of glycoproteins with special regard to periodic acid-schiff staining

Various investigators reported different normal values for the glycoprotein fractions obtained after electrophoretic separation of serum. The purpose of this investigation was to find the optimal conditions for each step of the periodic acid-Schiff staining and to determine standard deviations and normal values for the different glycoprotein fractions with the greatest possible accuracy.

The Application of Ultra-Thin Sectioning Techniques to Non-Biological Samples

Although ultra-thin sectioning techniques are widely used in the biological sciences, their applications are somewhat less popular but very useful in industrial applications. This presentation will review several specific applications where ultra-thin sectioning techniques have proven invaluable.The preparation of samples for sectioning usually involves embedding in an epoxy resin. Araldite 6005 Resin and Hardener are mixed so that the hardness of the embedding medium matches that of the sample to reduce any distortion of the sample during the sectioning process. No dehydration series are needed to prepare our usual samples for embedding, but some types require hardening and staining steps. The embedded samples are sectioned with either a prototype of a Porter-Blum Microtome or an LKB Ultrotome III. Both instruments are equipped with diamond knives.In the study of photographic film, the distribution of the developed silver particles through the layer is important to the image tone and/or scattering power. Also, the morphology of the developed silver is an important factor, and cross sections will show this structure.