Vital Staining by Trypan Blue; Its Selectivity for Olfactory Receptor Cells of the Brown Bullhead, Ictalurus Natalis

The affinity of olfactory receptors in fish for trypan blue as a vital stain is similar to that in amphibia and mammals. But, so far, only Ictalurus has given satisfactory staining. After anesthetizing the animals with MS 222 (tricaine methane sulfonate), the olfactory folds were laid bare by excising the flap of skin between anterior and posterior nares. Next, filter paper was employed to absorb the water and mucus between the folds which were then covered with staining solution. The variations in the treatment were: (a) exposure to 0.5, 1.0 and 2.0% stain concentration, (b) use of distilled water or of 0.7% NaCl solution, (c) staining times of 20, 40, 60 and 80 min. The treated tissues were fixed in Heidenhain's SUSA for 12 hr, dehydrated in isopropyl alcohol, and embedded by passage through methyl benzoate and benzene into paraffin. The sections were cut at 7 μ and mounted in Canada balsam. Vital staining in a 1% distilled water solution for 60 min gave the best results. The receptors were deep blue and contrasted well with the supporting cells. Also, the central receptor-cell processes had taken the stain. Preparations exposed longer than 60 min showed a loss of stain from the receptors. In Ictalurus all receptors are spindle-shaped with peripheral processes of little varying length and thickness depending on the depth of their nuclei within the epithelium. Most nuclei lie near the center of the epithelium. The central processes are thinner (0.25-0. 28 μ) and more curved than the peripheral ones (0.7-0. 9 μ) and may contain small pale blue apparently vesicular swellings. Trypan blue delimits receptor cells more sharply than silver impregnation and seems to be well suited for studies of comparative histology and cytology of vertebrate olfactory epithelium. Methylene blue vital staining of the same tissue shows selectivity for the intraepithelial bundles of central receptor processes rather than for the receptor nuclei.     

A triple-stain technique for evaluating normal acrosome reactions of human sperm

A triple-stain technique has been developed to score normal acrosome-reacted human sperm in fixed smears. Live and dead sperm are first differentiated using the vital stain trypan blue. Sperm are then fixed in glutaraldehyde, dried onto slides, and the postacrosomal region and acrosome are differentiated using Bismark brown and Rose Bengal. Slides are examined at 1,000 X with a bright-field microscope and assessed for 1) the percentage of sperm that were alive at the time of fixation and 2) the percentage of sperm that had undergone normal acrosome reactions. Experiments are included that show that trypan blue is a reliable stain for dead sperm and that Rose Bengal stains only sperm having intact acrosomes. This technique may have applications in experimental and clinical studies on sperm capacitation, acrosome reactions, and fertilization in laboratory and domestic animals as well as in man.     

Staining of Bacterial Colonies on the Millipore Filter to Improve Contrast

About 5 ml of 1% blue tetrazolium in 70% ethyl alcohol were poured over mature colonies of Pasteurella pestis and Malleomyces pseudomallei on Millipore filters (MF), contained in the filter holder apparatus, and allowed to drain through with the suction applied. The MF was washed with water and then covered with about 10 ml of 0.001% aqueous trypan blue and drained. This technique provided vivid white colonies sharply defined against a blue background.

Another method utilized 0.1% quinacrine-HCl (Atabrine) to stain colonies yellow and 0.05% vital red to stain the MF pink to light red.     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Activation of Mouse Oocytes, Fertilization and Development of Mouse Embryos in Vitro after Staining with Trypan Blue or Fluorescein Diacetate

The influence of vital staining with trypan blue or fluorescein diacetate on the fertilization of mouse oocytes and the developmental potential of mouse embryos was assessed. Neither stain induced spontaneous activation in mouse oocytes, nor did they impair the in vitro development and implantation of mouse zygotes, two-cell embryos, stressed morulae or blastocysts. However, fertilization and subsequent development of mouse oocytes have been shown to be reduced by vital staining.     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Combined Stain for Fish Pituitary

A new combined stain is described for the study of cell types in the fish pituitary. Tissues are prepared by fixing in formol-sublimate and then embedded in win wax. Tissue is sectioned at 5 μm and then stained sequentially with performic acid-alcian blue, periodic acid-Schiff, orange G, and acid fuchsin As a result of this procedure acidophils stain as follows: lactotropes, red; corticotropes, light pink melanotropes, bright pink and somatotropes, orange. Cyanophils stain either magenta red (gonadotropes) or blue (thyrotropes). Neurosecretory material and the fibers of the pars nervosa which penetrate the pars intermedia stain light blue.     

Combined stain for fish pituitary.

A new combined stain is described for the study of cell types in the fish pituitary. Tissues are prepared by fixing in formol-sublimate and then embedded in paraffin wax. Tissue is sectioned at 5 micron and than stained sequentially with performic acid-alcian blue, periodic acid-Schiff, orange G, and acid fuchsin. As a result of this procedure acidophils stain as follows: lactotropes, red; corticotropes, light pink; melanotropes, bright pink; and somatotropes, orange. Cyanophils stain either magenta red (gonadotropes) or blue (thyrotropes). Neurosecretory material and the fibers of the pars nervosa which penetrate the pars intermedia stain light blue.     

Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species

A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.     

Apoptosis and Morphology in Mouse Embryos by Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy combined with a vital stain was used to study apoptosis in organogenesis-stage mouse embryos. Apoptosis has previously been visualized in whole embryos using the vital dyes acridine orange, Nile blue sulfate, and neutral red. In the present study, mouse embryos were harvested on Gestation Day 9 and stained with the vital lysosomal dye LysoTracker Red. Following incubation in the stain, embryos were fixed overnight in 4% paraformaldehyde, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. To achieve optical sectioning through embryos, it was necessary to use low-power objectives. With this procedure, the entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. Our results demonstrate specific regions undergoing programmed cell death in normal development and increased LysoTracker staining in embryos exposed to hydroxyurea. This procedure allows for the optical imaging of whole Day 9 ( approximately 22 somites) embryos that were greater than 700 microm thick in the z axis and can be applied to studies involving neural tube formation or other aspects of organogenesis.     

4种染色方法对甜瓜白粉病菌染色效果的观察比较

考马斯亮蓝组织透明染色方法可以清楚观察到白粉病菌的5个发育阶段,即萌发的分生孢子、初生芽管、胞间菌丝、分生孢子梗和后期菌落。考马斯亮蓝几乎使寄主组织不着色,不产生背景色干扰,而菌体变深蓝色。苯胺蓝组织透明染色方法也可观察到病菌的不同发育阶段,但苯胺蓝易使寄主组织产生浅蓝色背景,而菌体呈现深蓝色,观察效果不理想。荧光素钠和苯胺蓝两种荧光染色方法均能使菌体在紫外或者蓝光下产生黄绿色荧光,而寄主组织呈现黑色背景,强烈的反差利于观察。荧光素钠可以观察到菌体整个发育阶段。苯胺蓝只适合于前期菌体入侵过程的观察。     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Some fluorescent counterstains for neuroanatomical studies

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.     

White gels: an easy way to preserve methylene blue stained gels

Methylene blue can be used as a stain for visualizing nucleic acids in polyacrylamide gel electrophoresis. However, its relatively low sensitivity and reversible binding make it a temporary stain that diffuses from the gel relatively fast. Here we describe a very simple method for fixing methylene blue bands in nucleic acid polyacrylamide gels. The procedure makes the methylene blue stain permanent and increases the visibility of the bands, also contributing to increasing the sensitivity of methylene blue.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Bolton-Hunter reagent as a vital stain for developing systems

The Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy, 5-[¹²⁵I]iodophenyl)propionate, was used as a vital stain for developing amphibian and tunicate embryos and for isolated cells (human erythrocytes and cultured chick limb mesenchymal cells). We found that the Bolton-Hunter reagent can be used on living cells at room temperature with techniques that are quite similar to the techniques routinely used to label isolated macromolecules in vitro. At concentrations of vital stain that were sufficient to label intracellular proteins in intact-cells, labeled cells underwent normal developmental sequences. Under these conditions, vital staining with the Bolton-Hunter reagent disproportionately labeled exterior proteins, and it seems likely that the Bolton-Hunter reagent is an especially good vital stain for cell surface and cell membrane proteins. The Bolton-Hunter stain is covalently bound, is not reutilizable, and appears not to disrupt natural physiological and developmental processes. Thus, we used the Bolton-Hunter reagent to follow the natural life spans of proteins in vivo and we were able to distinguish particularly long-lived proteins in Xenopus embryos.     

A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

A Multichromatic Stain for Lowicryl K4M Embedded Tissues

A staining procedure using celes-tine blue and chrome-trope 2R for Lowicryl K4M embedded tissues is presented. The stain produces a reliable multichromatic stain for light microscopy on Lowicryl embedded semithin sections.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.