Advantages of picrate fixation for staining polypeptides in polyacrylamide gels

When acetic acid-urea polyacrylamide gels with or without Triton X-100 were immersed in 0.1 M Na picrate, pH 7, to which 1/4 vol Coomassie blue staining solution (0.2% in 45% methanol, 10% acetic acid, 45% water) was added, proteins stained rapidly (within a few minutes in gels without Triton and within an hour in gels with Triton) with little or no background staining. Thus protein bands could be observed in a single step with no destaining. The picrate-Coomassie blue method fixed and stained a small peptide (bradykinin, nine amino acids) that was not observed in gels stained with fast green, silver, or Coomassie blue following fixation in 50% trichloroacetic acid. The picrate-Coomassie blue method gave high-contrast bands suitable for densitometry. Gels containing sodium dodecyl sulfate were also stained by the picrate-Coomassie blue method if they were first washed briefly (1 h) in 45% methanol, 10% acetic acid, 45% water, presumably to remove the detergent. These gels also stained rapidly with almost no background.     

Prefixing with Paradichlorobenzene to Facilitate Chromosome Study

A technic was developed which resulted in preparations containing many mitotic divisions with chromosomes well fixed and stained, rod-shaped, and spread throughout the cell. This technic has given good results with guayule (Parthenium argentatum), Crepis, Allium, Pisum, Lycopersicon, Tradescantia, and other plants. Material is prefixed in a saturated solution of paradichlorobenzene for 1-4 hours, fixed in 65% acetic acid (or other suitable fixative) for 12-24 hours, hydrolyzed in 10% HCl for 10-30 minutes at 60° C, rinsed in water, transferred to a drop of 45% acetic acid on a slide, and smeared and stained in aceto-orcein. The preparation may be made permanent by separating slide and cover glass in 1 part glacial acetic acid to 1 part absolute alcohol, putting them in absolute alcohol, and then recombining them with a drop of euparol.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

A Naphthol Yellow S and Erythrosin B Staining Procedure for Use in Studies of the Acrosome Reaction of Rabbit Spermatozoa

Rabbit spermatozoa suspended in Krebs-Ringer-phosphate containing 0.25% glucose were smeared on polylysine-coated slides and dried in air at room temperature for 2 hr to overnight. Smears were stained in 0.1% naphthol yellow S in 1.0% acetic acid for 30 min at room temperature, blotted, rinsed in 1.0% aqueous acetic acid for 10-15 sec, drained and stained for 7 min in a mixture of equal parts of aqueous naphthol yellow S and erythrosin B (final concentration of each dye 0.1% w/v) at pH 4.6-5.0 (pH adjusted with acetic acid). Stained slides were well rinsed in distilled water adjusted to pH 4.65.0 with acetic acid, blotted, allowed to dry completely, rinsed in xylene and mounted in synthetic resin. Acrosomal caps were stained cherry-red (apical ridge) to pink (dorsal and ventral aspects); postnuclear caps stained pale pink; nuclei were either unstained or stained a very faint yellowish-pink. The mid-piece and flagellum were stained different shades of pink. The procedure is simple, rapid, and gives highly reproducible results. When present, acrosomes are easily detected regardless of the density of the smear.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

A naphthol yellow S and erythrosin B staining procedure for use in studies of the acrosome reaction of rabbit spermatozoa.

Rabbit spermatozoa suspended in Krebs-Ringer-phosphate containing 0.25% glucose were smeared on polylysine-coated slides and dried in air at room temperature for 30 min at room temperature, blotted, rinsed in 1.0% aqueous acetic acid for 10-15 sec, drained and stained for 7 min in a mixture of equal parts of aqueous naphthol yellow S and erythrosin B (final concentration of each dye 0.1% w/v) at pH 4.6-5.0 (pH adjusted with acetic acid). Stained slides were well rinsed in distilled water adjusted to pH 4.6-5.0 with acetic acid, blotted, allowed to dry completely, rinsed in xylene and mounted in synthetic resin. Acrosomal caps were stained cherry-red (apical ridge) to pink (dorsal and ventral aspects); postnuclear caps stained pale pink; nuclei were either unstained or stained a very faint yellowish-pink. The mid-piece and flagellum were stained different shades of pink. The procedure is simple, rapid, and gives highly reproducible results. When present, acrosomes are easily detected regardless of the density of the smear.     

Acetic Acid as a Diluent and Dehydrant in the Preparation of Whole, Stained Helminths

Aqueous 45% acetic acid can be used successfully as a diluent for Ehrlich's haematoxylin and for Horen's trichrome stain (chromotrope 2 R, 0.6 gm; phosphotungstic acid, 0.7 gm; glacial acetic acid, 1.0 ml; water, 100 ml). Glacial acetic acid is used for dehydration of the stained helminths, and followed by a glacial acetic acid-methyl salicylate series for clearing. The whole process can be completed within 1 hr, from fixation to the cleared specimen, with helminths up to 5 mm in length. A satisfactory fixative for Monogenea, Digenea and Acanthocephala is: 85% ethanol, 85; formalin (40% HCHO), 10; and glacial acetic acid, 5—parts by volume. For Cestoda, 5% aqueous formalin is preferable because they are hardened excessively by the alcoholic fixative.     

Azophloxine in Two-Dye and Four-Dye Staining Combination

Tissues are fixed in either 10% formalin or Lavdow-sky's mixture. After the tissues are sectioned and mounted, they are stained in hematoxylin, then counterstained for 2 minutes in 0.1% aqueous azophloxine to which 4 drops of acetic acid have been added to each 100 ml. of stain. Sections are then rinsed in 0.2% acetic acid and dehydrated. Azophloxine GA can be used also in a tetrachrome method. Sections are stained in Harris' hematoxylin, washed, and placed in 0.2% acidified aqueous azophloxine for 2 minutes. They are then rinsed in 0.2% acetic acid, stained 1 minute in an aqueous mixture of 4% phosphotungstic acid and 2% orange G solution and rinsed again in 0.2% acetic acid. Finally, they are stained in 0.2% light green for 2 minutes, and differentiated in 0.2% acetic acid for 5 minutes. The advantage in using azophloxine is that it is clear and delicate and when used in a constant concentration, does not overstain if the recommended procedure is followed.     

Trypan Blue as a Stain for Fungi

Trypan blue was tested for use in the study of fungi. It was found that it stained the cell wall of all fungi investigated. Two staining solutions were very satisfactory: (1) A quick-staining solution of 0.1-0.5% dye in 45% acetic acid; (2) A slow-staining solution of 0.1-0.5% dye in lactophenol. In both instances, heating the preparations quickened the action.     

A Combined Hcl-Acetic-Alcohol Fixation and Hydrolysis Followed by Cresyl Violet Staining for Mosquito Chromosome Spreads

Mosquito tissues of cytogenetical importance were dissected out on a slide in 0.65% NaCl, under a dissecting microscope, and treated about 30 sec in a drop of 1:3 Carnoy's fixative diluted 1:19 with distilled water. Fixing and hydrolysis was done by a single step in a mixture consisting of: glacial acetic acid, 1; ethanol 96%, 3; HCl conc., 2; and distilled water, 2 (v/v) for 2-6 min at 20-25 C. The specimen was then rinsed with the acetic-alcohol fixative and covered in a drop of 1% cresyl violet in 50% acetic acid under a coverslip coated with Mayer's albumen. Washing was performed immediately by adding water dropwise to one side of the coverslip and drawing the fluid from the other side with absorbent paper. The preparation could be used either as a temporary slide or made into a durable mount. The DNA-containing bands of the giant polytenic chromosomes stained dark violet; interband regions, weakly stained or colourless against a clear background. Mitotic and meiotic figures in gonadal cells stained selectively dark violet or violet with a practically unstained cytoplasm.     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

Aceto-Carmine-Lactophenol Preparations Of Paramecium Aurelia

Paramecia were killed and stained by adding a sat. soln. of carmine in acetic acid to a small drop of culture, cleared with 45% acetic acid as soon as the nuclei became darkly colored, and mounted in lactophenol (phenol crystals, 20 g.; lactic acid, 20 ml.; glycerol, 40 ml.; distilled water, 20 ml.). The mounting mixture was prepared by warming to dissolve the phenol and 20 drops of aceto-carmine added after cooling. Cover glasses were ringed with colorless nail polish or with asphaltum after slides had stood several days.     

An improved method for preparing whole specimens from bovine preimplantation embryos: A technique note

A method was devised to prevent loss of whole embryos during fixation. Specimens were prepared in a chamber saturated with fixative vapors consisting of 3 : 1 (v/v) 96%. ethanol/glacial acetic acid. Good quality specimens were obtained after fixation for at least 24 but not more than 72 h. After staining, specimens could be preserved for 3 to 4 d by storage in the fixation chamber, in 45% aqueous acetic acid vapor. Using the method suggested in this paper prevents loss of early embryos during fixation and allows storage of specimens for longer than usual time while maintaining the quality of the specimen.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

A new method for the preparation of band 3, the main integral protein of the human erythrocyte membrane

Band-3 protein from human erythrocyte membranes was isolated, without using detergents, by a two-step procedure: (1) The peripheral proteins were removed from the membrane by treatment with 10% acetic acid. (2) The remaining lipoprotein complex was solubilized in approximately 92% (v/v) acetic acid and then separated into its components by preparative zonal electrophoresis in a gradient made up of acetic acid, water and sucrose. Band 3 was recovered from the gradient at a yield of 60 - 70% and purity of about 95%. Approximately 25 mg of band 3 could be prepared in one run. The protein is soluble in aqueous solutions, even in the absence of organic solvents or detergents. In addition to band 3, the proteins stained by periodic acid/Schiff's reagent (the sialoglycoproteins) are also separated from the other proteins.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine, Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine,Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.