Improved Movat Pentachrome Stain

Movat in 1955 developed a staining method which demonstrates collagen fibers, mucin, muscle fibers, elastic fibers, fibrin and fibrinoid changes in a single section. His procedure was considered excellent by Lynch et al. (1969)     

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [³H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [³H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

Rapid Identification of Chromosomal Abnormalities in Human Neoplasia

Recent advances in banding techniques have led to the belief that certain chromosomal defects are consistently associated with specific types of human neoplasia. Based on the GTG technique, it has been suggested that the malignant cells of most neoplasias show chromosomal abnormalities (Yunis et al. 1983). From this recent publication of Yunis it appears that the majority of bands involved in carcinogenesis are G-negative, i.e., do not stain by the GTG technique, and it is therefore difficult to localize the breakpoints. In some of our recent publications we emphasized the importance of the RFA technique (Verma and Lubs 1975), which stains Giemsa-negative bands darkly, thus providing precise identification of chromosomal abnormalities (Verma and Dosik 1976). However, this technique cannot be applied until the slides have aged for at least 7 days. Therefore, we are reporting an alternative procedure using BrdU which provides “reverse” banding immediately when the slides are stained with acridness orange and examined with a fluorescence microscope.     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

A Quick and Standardized Giemsa Stain for Wet-Fixed Cytological Material

The Giemsa stain is one of the most widely used staining techniques in cytology, especially in hematology. A standardized Romanowsky-Giemsa staining procedure using pure cationic azure B (C.I. 52010) and anionic eosin (C.I. 45380) has been described by Wittekind et al (1982). A revised standard Giemsa staining procedure was recently published (Wittekind and Kretschmer 1987). Usually the Romanowsky-Giemsa stain is applied to air dried and methanol fixed cytological material, e.g. blood smears and bone marrow films (ICSH 1984).     

Improvement in the Specificity of the Silver Staining Technique for Ag NOR-ASSOCIATED Acidic Proteins in Paraffin Sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiffs reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

Fluorescent Staining of Juxtaglomerular Cells in Frozen Sections

Enzymatic investigations of the juxtaglomerular apparatus often creates the need for visualisation of granulated juxtaglomerular cells (JGC) in preparations subjected to histochemical procedures. In our investigations, Pitcock and Hartroft's (1958) modification of Bowie's method and the Endes et al. (1969) combined trichrome staining proved to be inadequate when applied to fresh cryostat sections, or to formol- or glutaraldehyde-fixetl, gum sucrose-impregnated frozen sections. Friedberg and Reid's (1966) crystal violet procedure for waxembedded kidneys also failed to give uniformly reproducible results. In attempting to find a satisfactory technique for both enzyme and granule staining, we noted Janigan's (1965) and Haratla's (1969) observations on paraffin-embedded JGC, and tested the following fluorochromes: thioflavine T—Fluka, C. I. 49005; auramine O—Merck, C. I. 41000; acridine orange—E. Gurr, C. I. 46005; berberine sulfate—Fluka, C. I. 75160 on 10 μ sections of albino mouse kidneys prepared in 4 different ways as follows:     

Improved Stability of A Purified Glycol Methacrylate Preparation: Comments

The technical note from Bernier et al. (1984) presents additional observations on our procedure for purifying glycol methacrylate (GMA), a hydrophylic resin (Chappard et al. 1982). It is becoming increasingly popular and widely used as an embedding medium for light microscopic studies. GMA is prepared by esterification of methacrylic acid (MA), but about 1% of free unreacted MA remains in the monomer. MA can copolymerize with GMA and it also binds strongly to thiazin and other basic dyes (Tipett and O'Brien 1975) so as an undesirable impurity it must be removed.     

Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Signalling via rat dopamine D2-receptors expressed in mouse fibroblasts is not influenced by compounds binding to the sigma sites of these cells

The mouse fibroblast cell line LZR-1 is a well-established test system used to characterize the intrinsic activity of dopamine D₂-receptor ligands (Neve et al., Mol. Pharmac., 1989). This cell line is transfected with a eucaryotic expression vector containing the gene of the rat dopamine D_2B-receptor (short isoform of the D₂-receptor) (Bunzow et al., Nature, 1988). In addition to the expression of high levels of the rat dopamine D₂-receptor, these mouse cells also express sigma binding sites. Binding affinities of BMY 14,802, DTG, haloperidol, ( + )-pentazocine, ( + )-3-PPP, and ( + )-SKF 10,047 for the sigma binding sites as well as for the D₂-dopamine receptor in membranes of LZR-1 cells were determined. Using intact LZR-1 cells, it was found that the influence of sigma ligands on signalling via the dopamine D₂-receptor can be explained by their affinity for the latter receptor. Specific sigma ligands did not influence dopaminergic signal transduction in LZR-1 cells. It is therefore concluded that the LZR-1 cell is a suitable test model for determination of the intrinsic activity of dopamine D₂-receptor ligands even if these compounds have affinity for sigma binding sites.     

Notes on Technic: Simple, Reliable Detection of Latex Microspheres in High Quality Tissue Sections

Latex microspheres used in biological research have been visualized by light microscopy in mounts of cell suspensions, disrupted cells, or cleared tissues (Mishima et al 1987, Koonce et al 1986, LeFevre et al 1978); in unembedded coverslip monolayers (Koerten et al 1980); in fixed (Cornwall and Phillipson 1988) or unfixed (Wells et al 1988) frozen sections; in paraffin sections cleared and deparaffinized with n-butyl alcohol (Callebaut and Meeussen 1989); and in tissues embedded in resins suitable for transmission electron microscopy, such as Spurr's (Hampton et al 1987), Epon (Herzog and Miller 1979), or Ladd Low Viscosity Epon (LeFevre et al 1985). Paraffin embedding, and some plastic embedments, are impractical for demonstration of latex beads because the beads are dissolved by such organic solvents as xylene, dioxane, or chloroform (Van Furth and Diesselhoff-Den Dulk 1980), propylene oxide (Lentzen et al 1984), amyl acetate (Okada et al 1981), or toluene, the solvent in commonly used mounting media such as Fisher Permount (personal observation). The space remaining after dissolution of a bead is not maintained with paraffin embedding as it is with resin embedding. Even after plastic embedding, the resolving power of light microscopes may be inadequate to distinguish such spaces from other spaces found in and between cells. Latex beads are stable in methanol (Van Furth and Diesselhoff-Den Dulk 1980), ethanol. and n-butyl alcohol (Callebaut and Meeussen 1989).     

Modification of the Carmen-Faull-Pullar Racks for Free-Floating Serial Sections

The original staining racks designed by Carmen et al. (Stain Techn., 43: 157-60) have been redesigned to use inside plates of 0.06 inch thickness and outside (top and bottom) ones of 3/16 inch. The greater thicknesses permit freer circulation about the sections and avoid the need for outer clamps to hold the assembly together. As in the original racks acrylic sheeting has been used, but with 25 × 45 mm holes for sections in each plate. Ordinary fiberglass window screening was cemented to one side of each plate. The assembly of 12 inner and the 2 outer plates was held together by 2 bolts made of 1/4 inch acrylic rod. Since clamps on the edges of the assembly were not needed, smaller staining dishes could be used, with coincident economy in volume of staining solutions.     

Combined Staining by Thiolacetic Acid and Bielschowsky Methods

Tongues of mice were fixed in 10% Baker's Ca-formalin and sectioned at 50 μ with a freezing microtome. The thiolacetic acid method (Wachstein et al., J. Histochem. Cytochem., 9: 325-39) was used for motor endplates and after this cholinesterase staining, the Bielschowsky method was applied for nerve fibers. Thus, both motor endplates and nerve fibers were fully demonstrated.     

微生物新型防御系统的系统性发现与展望

微生物防御系统是生物技术创新与发展的重要工具库,细菌限制性内切酶的发现催生了现代分子克隆技术,CRISPR系统的开发利用则使基因组编辑技术取得革命性突破。基于上述原因,微生物新型防御系统的发现与研究已引起各国科学家的重视,一些新的防御系统如pAgos和DISARM等相继被发现和研究。为进一步挖掘微生物中可能蕴藏着的其他未知防御系统,最近以色列科学家Sorek等报道了从海量的微生物基因组序列中系统性发现新型防御系统的研究策略,并且通过合成生物学思路鉴定了10种新型系统的抗病毒或抗质粒功能。本文将首先介绍Sorek团队系统性发现新型防御系统的研究工作,进而总结目前已知的主要微生物新型防御系统的可能机制,并对该领域的发展态势与挑战进行分析和展望。     

Endpoint error in smoothing and differentiating raw kinematic data: An evaluation of four popular methods

‘Endpoint error’ describes the erratic behavior at the beginning and end of the computed acceleration data which is commonly observed after smoothing and differentiating raw displacement data. To evaluate endpoint error produced by four popular smoothing and differentiating techniques, Lanshammar's (1982, J. Biomechanics 15, 99–105) modification of the Pezzack et al. (1977, J. Biomechanics, 10, 377–382) raw angular displacement data set was truncated at three different locations corresponding to the major peaks in the criterion acceleration curve. Also, for each data subset, three padding conditions were applied. Each data subset was smoothed and differentiated using the Butterworth digital filter, cubic spline, quintic spline, and Fourier series to obtain acceleration values. RMS residual errors were calculated between the computed and criterion accelerations in the endpoint regions. Although no method completely eliminated endpoint error, the results demonstrated clear superiority of the quintic spline over the other three methods in producing accurate acceleration values close to the endpoints of the modified Pezzack et al. (1977) data set. In fact, the quintic spline performed best with non-padded data (cumulative error=48.0 rad s⁻²). Conversely, when applied to non-padded data, the Butterworth digital filter produced wildly deviating values beginning more than the 10 points from the terminal data point (cumulative error=226.6 rad s⁻²). Each of the four methods performed better when applied to data subsets padded by linear extrapolation (average cumulative error=68.8 rad s⁻²) than when applied to analogous subsets padded by reflection (average cumulative error=86.1 rad s⁻²).     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Embedding Free-Floating Cells in Stained Agar for Rapid Scanning and Subsequent Ultramicrotomy

Methods we have previously used for epoxy embedding of suspended tissue culture cells, white blood cells and oral mucosal scrapings did not provide a simple means for selecting the desired specimens and facilitating their subsequent handling through embedding in an epoxy resin. We have therefore modified the agar embedding technique previously recommended for the processing of microorganisms by Kellenberger et al. (1958).     

Demonstration of the Microvasculature of the Spinal Cord by Intravenous Injection of the Fluorescent Dye, Thioflavine S

In the past, thioflavine S has been used for visualizing blood vessels and patterns of blood flow (Schlegel 1949; Schlegel and Moses 1950; Oliver et al. 1951). Methods employed have involved an intravenous injection of the dye, immersion of hand-cut sections in glycerol and examination of sections under incident Wood's light. With improved techniques it is possible to obtain microtome-cut sections and to use a more intense light source for enhancing fluorescence and resulting visualization of small vessels. Occlusion of arterioles by undissolved dye particles is prevented by ultracentrifugation of the solution to be injected.