The Role of Inhibitors in the Fluorescent Staining of Benign Naevus and Malignant Melanoma Cells with 9-Amino Acridine and Acridine Orange

Abstract

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

Staining of Small Lymphoid Nucleoli in Paraffin Sections by Aniline-Azure B

To see small lymphoid nucleoli clearly in 1-2 μ paraffin sections, the staining of contiguous chromatin masses in the nucleus was suppressed by a hydrolysis-aniline blocking sequence, which produces aldehyde from DNA, and attaches aniline to that aldehyde to make a diphenamine base, thus reducing the acidity of the chromatin and its affinity for basic dyes. Nucleolar RNA remains fully stainable by azure B, because the hydrolysis used does not produce aldehyde groups in it, to allow aniline attachment. Technique: Hydrolyse the 10% formol-saline fixed, deparaffinised 1-2 μ section for 4.5-5.0 min in 10% (v/v) HCl in tetra-hydrofuran at 39-40 C, rinse in water, and treat at room temperature in 10% (v/v) aniline in acetic acid for 10 min. Stain 2-4 hr with freshly prepared 0.1% azure B in a 1:10 dilution of tris buffer at pH 7.0. Rinse, blot off excess water, pass through acetone and xylene to a polystyrene mounting. DNA stains pale green to colourless; nucleolar and cytoplasmic RNA, blue.     

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.     

Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Sequential Estimation of Nuclear DNA and Silver Staining of Nucleoli in Plant Cells

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO₃. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G_I phase of the cell cycle.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

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:     

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light-catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.     

Methyl Green as a Cartilag Setain; Human Embryos

The development of this technique derived from a need to demonstrate the sites of developing cartilage in the human embryo. The basic procedure was that of Yntema (1970), which used methyl green on turtle embryos. However, the thickness of the soft tissues in human embryos of 90-125 mm crown-rump (CR) length and the opacity caused by pigments in this tissue when the specimen was cleared necessitated the modification of the original procedure; specifically, the introduction of bleaching. The altered procedure is given below.     

Enhancing Aniline Blue Fluorescent Staining of Cell Wall Structures

Prior staining with the periodic acid-Schiff reaction, toluidine blue O, Congo red, or Calcofluor White M2R New, or reduction by NaBH₄ do not interfere with aniline blue-induced fluorescence of sieve plates, new cell walls, pit fields or tracheids in compression wood of conifers. Detail of such fluorescent structures is improved by these treatments because of increased contrast, reduced flare, and a quenching of autofluorescence.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...     

Vital Fluorescent Staining Technique for Microspores of Brassica Napus

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus.It provided high contrast when used in combination with Tinapol 5BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.     

Acridine Orange Staining and Fluorescence of Nucleic Acids in Plasmodia and Associated Host Erythrocytes

Acridine orange in daily doses of 1, 2 and 4 mg for 4 days was given to chicks averaging 50 gm in weight. Dosage was started 1, 2 and 3 days after infection with Plasmodium gallinaceum. Such doses were sufficient to stain the parasite in vivo, as shown by its bright fluorescence in UV light, but did not exhibit any antimalarial action. Staining of fresh blood samples from infected chicks with 0.01% acridine orange in Krebs-Ringer containing 0.1 M phosphate buffer (pH 6.0-6.2) resulted in differential fluorescence of the nucleic acids of the plasmodia, to show nuclear DNA bright green and cytoplasmic RNA orange-red. After optimum acid hydrolysis, as used for the Feulgen reaction, staining with 0.1% acridine orange produced intense red fluorescence of the nuclear DNA in the plasmodia. Nuclear DNA of the chick erythrocytes showed bright fluorescence both in vivo and in vitro.     

Methyl Green-Pyronin for Staining Autoradiographs of Hydroxyethyl Methacrylate-Embedded Lymphoid Tissue

Cells in the spleen in DNA-synthesis were labelled with tritiated thymidine. Tissue was fixed for 12 hr in 10% neutral formalin, washed for 4 hr in tap water and dehydrated through 70% and absolute ethanol. The tissue blocks were infiltrated overnight with a mixture consisting of glycol methacrylate, 80 ml; polyethylene glycol 400, 12 ml; and benzoyl peroxide, 0.27 gm. Specimens were cast in BEEM capsules with the final embedding medium consisting of 42 parts of the infiltration medium and 1 part of an acceleration mixture. This mixture consisted of N,N-dimethylaniline, 1 part and polyethylene glycol 400, 15 parts. The blocks hardened in 30 min and were sectioned with an ultramicrotome fitted with glass knives. Sections were coated with Ilford K5 liquid emulsion and exposed for 2 wk. Methyl green-pyronin staining of autoradiographs was carried out at pH 4.1 in acetate buffer containing 0.5% methyl green (Allied Chemicals) and 0.2% pyronin GS (Chroma). Staining was for 30-60 min, after which sections were washed for 1 min in water, blotted, allowed to dry, and mounted in Canada balsm. The procedure resulted in good quality autoradiographs in which the degree of basophilia of labelled cells could be assessed.     

Variants of Green Fluorescent Protein GFPxm

As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could produce strong fluorescence when expressed at 37°C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37°C, and mutants GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42°C. Meanwhile, at least 4 mutants could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak.