Cytochemical Method to Localize Acidic Nuclear Proteins

Fast green FCF was used to localize acidic nuclear proteins in sections of young flower buds of Limnophyton obtusifolium (L.) Miq. After extracting nucleic acids, the slides were stained at hydrogen ion concentrations ranging from pH 2.6 to 9.0. At pH 5.0 and 8.0 staining is confined to the nucleus with no cytoplasmic reaction. Staining intensity is greater at pH 5.0 than at pH 8.0. The proteins responding to fast green at pH 8.0 are basic proteins. The positive reaction at pH 5.0 is attributed to acidic nuclear proteins. These findings are confirmed by control preparations. Acetylated slides and slides treated with 0.25 N HCl were unstained at pH 8.0 but staining at pH 5.0 was undisturbed. Dilute alkali (0.003 N NaOH) reduced staining intensity at pH 5.0 but had no effect at pH 8.0. Methylated slides did not stain at pH 5.0, but at pH 8.0 staining was unaffected. Deamination blocked staining at both pH's. It is concluded that fast green at pH 5.0 specifically binds with acidic nuclear proteins.     

The Use of Buffered Solutions in Staining: Theory and Practice

The importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl₃, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.     

AN EMPIRICAL PROTOCOL FOR WHOLE-CELL IMMUNOFLUORESCENCE OF MARINE PHYTOPLANKTON1

An immunofluorescence protocol for intracellular antigens of phytoplankton was developed empirically. Paraformaldehyde (4%) was used to fix samples for 6 h at 4° C; fixed samples can be stored in cold (–20°C) methanol for at least 1 month. We used Triton X-100, Nonidet P-40, and dimethyl sulfoxide to permeabilize the cells for antibody penetration across the cell wall and plasma membrane. After immunolabeling at room temperature, the samples were mounted on slides and could be stored at 4° C for up to 6 months without significant decay of the fluorescence. The staining was examined with an epifluorescence microscope; semiquantitative (percentage of cells with positive staining) and quantitative (staining intensity measured with an imaging system) analyses were performed. Reproducible staining was achieved for nuclear, chloroplastic, cytoplasmic, and cell surface antigens in species of Chlorophyceae, Prymnesiophyceae, Bacillariophyceae, and Dinophyceae including some field-collected samples. This protocol is advantageous to previously published protocols in that it allows relatively simple and long-term storage of fixed samples and allows staining of multiple antigens for the same sample. Although improvement on cell permeabilization is required for some species, the present protocol could prove useful for physiological or ecological studies when frequent sampling is required and immediate processing of the samples is not possible.     

Improved Coomassie Blue Dye-Based Fast Staining Protocol for Proteins Separated by SDS-PAGE

The time required to visualize proteins using Coomassie Blue dye has been significantly reduced with the introduction of fast staining protocols based on staining with a Coomassie Blue dye solution at boiling temperatures. However, fast stainings suffer from high gel backgrounds, reducing the signal-to-noise ratio and limiting the number of detectable spots in the case of 2D SDS-PAGE. The aim of this work was to eliminate the high gel background, and thus improve fast staining protocols based on Coomassie Blue dye. We show that merely replacing water with a 4 mM EDTA washing solution at boiling temperatures, results in a transparent gel background within 50 to 60 minutes of destaining. Moreover, when a combination of imidazole-zinc reverse staining and Coomassie Blue-based fast staining is used the sensitivity is improved significantly; nanogram amounts of proteins can be detected using 1D SDS-PAGE, and about 30% to 60% more spots can be detected with 2D SDS-PAGE in plasma, platelet, and rat brain tissue samples. This work represents an optimized fast staining protocol with improved sensitivity, requiring between 60 to 75 minutes to complete protein visualization.     

About the mechanism of interference of silver staining with peptide mass spectrometry

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.     

Acridine orange binding to chromatin of individual cells and nuclei under different staining conditions. Thermadenaturation of Chromatin.

The thermodenaturation of chromatin in situ was studied by staining heat-treated nuclei with acridine orange. It was found that formaldehyde, which under the present conditions had to be used to prevent extensive renaturation of DNA, seriously affects the results of standard acridine orange staining in an unspecific manner. In particular the acetylation step involved in this staining method is strongly inhibited. Thus the standard method of staining can only give qualitative information about the effects of thermodenaturation. On the other hand, acridine orange staining at defined equilibrium, without prior acetylation is insensitive to formaldehyde and multiphasic thermodenaturation profiles are obtained with this method. At low temperatures these profiles mainly reflect changes in the protein-DNA interaction whereas at higher temperatures DNA denaturation also contributes to the curves. Although these two processes cannot be separately quantitated by simple measurements of dye binding, the thermodenaturation profiles still contain biologically significant information about the properties of chromatin in situ.     

Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.     

A new version of the Ag?NOR technique. A combination with DAPI staining

Summary The Ag−NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag−NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at −20°C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag−NOR staining was determind experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag−NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Gram Staining Applied to Human Spermatozoa: a Simple Method for Studying Chromatin Condensation Status

Gram staining applied to human spermatozoa from fertile donors is described. The stain revealed populations of Gram-positive and Gram-negative spermatozoa. Data showed a significant and progressive decrease in the percentage of Gram-positive spermatozoa at different times during the chromatin decondensation procedure (SDS-BSA and SDS-EDTA). No significant correlation could be found between Gram staining and other functional tests used for spermatozoa; only the aniline blue staining test showed a poor correlation. Our study demonstrates that normal spermatozoa with regular chromatin condensation appear Gram-positive, while spermatozoa with altered chromatin condensation appear Gramnegative.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

An optimized device for staining electron microscopy grids

Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 μl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.     

An immunoelectron microscope study of the organization of proteoglycan monomer, link protein, and collagen in the matrix of articular cartilage

Monospecific antibodies to bovine cartilage proteoglycan monomer (PG) and link protein (LP) have been used with immunoperoxidase electron microscopy to study the distribution and organization of these molecules in bovine articular cartilage. The following observations were made: (a) The interterritorial matrix of the deep zone contained discrete interfibrillar particulate staining for PG and LP. This particulate staining, which was linked by faint bands of staining (for PG) or filaments (for LP), was spaced at 75- to 80-nm intervals. On collagen fibrils PG was also detected as particulate staining spaced at regular intervals (72 nm), corresponding to the periodicity of collagen cross-banding. The interfibrillar PG staining was often linked to the fibrillar PG staining by the same bands or filaments. The latter were cleaved by a proteinase-free Streptomyces hyaluronidase with the removal of much of the interfibrillar lattice. Since this enzyme has a specificity for hyaluronic acid, the observations indicate that the lattice contains a backbone of hyaluronic acid (which appeared as banded or filamentous staining) to which is attached LP and PG, the latter collapsing when the tissue is fixed, reacted with antibodies, and prepared for electron microscopy. Thishyaluronic acid is anchored to collagen fibrils at regular intervals where PG is detected on collagen. PG and LP detected by antibody in the interterritorial zones are essentially fully extractible with 4 M guanidine hydrochloride. These observations indicated that interfibrillar PG and LP is aggregated with HA in this zone. (b) The remainder of the cartilage matrix had a completely different organization of PG and LP. There was no evidence of a similar latticework based on hyaluronic acid. Instead, smaller more closely packed particulate staining for PG was seen everywhere irregularly distributed over and close to collagen fibrils. LP was almost undetectable in the territorial matrix of the deep zone, as observed previously. In the middle and superficial zones, stronger semiparticulate staining for LP was distributed over collagen fibrils. (c) In the superficial zone, reaction product for PG was distributed evenly on collagen fibrils as diffuse staining and also irregularly as particulate staining. LP was observed as semiparticulate staining over collagen fibrils. The diffuse staining for PG remained after extraction with 4 M guanidine hydrochloride. (d) In pericellular matrix, most clearly identified in middle and deep zones, the nature and organization of reaction product for PG and LP were similar to those observed in the territorial matrix, except that LP and PG were more strongly stained and amorphous staining for both components was also observed. (e) This study demonstrates striking regional variations of ultrastructural organization of PG and LP in articular cartilage...     

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.     

Biotinylated theta-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.