Ciliary microtubule capping structures contain a mammalian kinetochore antigen

Structures that cap the plus ends of microtubules may be involved in the regulation of their assembly and disassembly. Growing and disassembling microtubules in the mitotic apparatus are capped by kinetochores and ciliary and flagellar microtubules are capped by the central microtubule cap and distal filaments. To compare the ciliary caps with kinetochores, isolated Tetrahymena cilia were stained with CREST (Calcinosis/phenomenon esophageal dysmotility, sclerodactyly, telangiectasia) antisera known to stain kinetochores. Immunofluorescence microscopy revealed that a CREST antiserum stained the distal tips of cilia that contained capping structures but did not stain axonemes that lacked capping structures. Both Coomassie blue-stained gels and Western blots probed with CREST antiserum revealed that a 97-kD antigen copurifies with the capping structures. Affinity-purified antibodies to the 97-kD ciliary protein stained the tips of cap-containing Tetrahymena cilia and the kinetochores in HeLa, Chinese hamster ovary, and Indian muntjak cells. These results suggest that at least one polypeptide found in the kinetochore is present in ciliary microtubule capping structures and that there may be a structural and/or functional homology between these structures that cap the plus ends of microtubules.     

Venetian Turpentine as an Aid in Squashing and Concomitant Production of Durable Chromosome Mounts

Root tips are hydrolyzed in 1 N HCl at 60 C for 10-12 min, Feulgen stained, and macerated in a minimal amount of propiono-carmine. A drop of Venetian turpentine mounting medium (Harleco brand was used) is mixed with the carmine stain, the cover slip applied, the tissue pressed gently while observed under a dissecting microscope to spread the chromosomes, and finally firmly squashed. High quality slides of over 1 yr durability are obtained which are well suited to morphological studies, photographing under oil, or routine screening in polyploidy studies. The carmine stain in conjunction with the Feulgen aids to give contrast to chromosomes which are difficult to stain (e.g. hops) but it may be omitted for other species.     

Coumarin in Chromosome Analysis

Root tips of monocotyledons were soaked 2.5-3.0 hours at 25-27° C. in saturated aqueous coumarin solution and stained in a mixture of N HC 1 and 2% aceto-orcein (1:9 by volume) 3-4 seconds over a flame. They were then squashed in 1% orcein under a cover glass, the excess stain blotted and the cover sealed. Preparations could be kept about one week. Good chromosome morphology was secured.     

The Demonstration of Beryllium Oxide in Paraffin Sections with Chromoxane Stains

Aqueous solutions of the arylmethane dyes Chromoxane pure blue BLD (C.I. No. 43825) and Chromoxane pure blue B (C.I. No. 43830) will stain beryllium oxide. In the presence of EDTA the staining of other metals is masked. As a specific stain for BeO, formol saline fixed paraffin sections are hydrated and stained for 1 hr with either 0.1 gm of pure blue BLD in 100 ml of pH 4.0 Na-acetate buffer or with 0.1 gm of pure blue B in 1 N NaOH adjusted to pH 9.0 with HCl. To mask interference from other metal ions, 9 gm of Na₂-EDTA is added to 100 ml of the stain solution. BeO is stained blue, organic tissue components are either unstained or pink. Results of tests against other materials show that a high degree of specificity may be expected from these dyes. A 1% aqueous solution of neutral red may be used as a counterstain.     

Staining and Washing Ultrathin Sections; a Device for Handling Various Materials Simultaneously

To eliminate individual manipulation, as many as 10 grids, each held firmly by a small notched bar of polyethylene plastic, are simultaneously stained, then washed. If the stain used is reactive with atmospheric CO₂ it can be forced through a Millipore filter into a small chamber made of glass tubing which contains the grid holder. The stain, cleared of any solid particles, has very little contact with air and remains free of lead carbonate contamination. Washing is carried out by submerging the chamber and removing the grid holder under water (Feldman, D. G., J. Cell Biol., 15: 592-5, 1962). Washing is minimized because there is not the risk of contaminating grids and wash water with stain trapped between the points of forceps. The polyethylene is nonadherent to the wash water, and the grids can therefore be dried quickly on the holder. With this method, the relative stainability of different materials may be observed because each grid within a batch receives identical treatment.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

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.     

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

THE USE OF BISMUTH AS AN ELECTRON STAIN FOR NUCLEIC ACIDS

Evidence is presented to show that bismuth combines in vitro with the phosphate of nucleic acids in a manner similar to its reaction with inorganic phosphate. When tested under similar conditions, protein exhibited no attraction for bismuth. The results of the in vitro experiments, which are of interest within themselves, may be indirectly applicable to in vivo staining. Dividing cells of onion root tips were fixed in OsO₄, stained with bismuth, and examined in the electron microscope. The electron opacity of cell structures known to contain nucleic acids was enhanced by bismuth, while organelles known to lack appreciable quantities of DNA or RNA showed little, if any, change. Bismuth is particularly effective as a stain for the chromatin material during interphase and for the chromosomes during division.     

Neuron Staining by Basic Dyes Versus Basic Metal-Dye Complexes; Differences Shown by Histochemical Blocking Reactions

Various blocking procedures were applied to sections of paraffin-embedded, formalin-fixed cat spinal cord. Treated sections and untreated controls were stained with cresyl violet acetate or gallocyanine-chrome alum. Although both dyes have been said to stain by simple salt formation it was found that staining was affected differently for each dye by the blocking procedures, and also that staining of neuron nuclei differed in the controls. In these, the cresyl violet acetate stained only the nucleoli within the nucleoplasm whereas gallocyanine-chrome alum stained much more material of unknown composition and function. It is proposed that if cresyl violet acetate and other basic dyes stain by salt linkage, and can be specific for nucleic acid and other highly acid materials, then gallocyanine and other basic metal dye complexes can not be specific for nucleic acid and do not stain by a simple salt linkage.     

THE ENTERIC SURFACE COAT ON CAT INTESTINAL MICROVILLI

The enteric microvilli of the cat, bat, and man are coated with a conspicuous layer composed of fine filaments radiating from the outer dense leaflet of the plasma membrane. This surface coat is prominent on the absorptive cells but is not so thick on the goblet and undifferentiated crypt cells. In other species the surface coat is poorly developed or inconsistent, but all intestinal microvilli have traces of such a coating over the tips and sides of the microvilli. Tissues prepared by the ordinary sectioning techniques for electron microscopy usually reveal this component when stained with uranyl acetate followed by lead staining. The surface coat is intensely periodic acid-Schiff (PAS) positive and reacts with Alcian blue or Hale's colloidal iron stain for acid mucopolysaccharide. It is also stained by toluidine blue at low pH. Repeated washings or incubation with various chemical agents have failed to remove or markedly alter the appearance of the coating, but extruded cells undergoing autolysis lose their surface coats. The stability, consistent presence, and intimate association of the mucopolysaccharide coat suggest that it may be an integral part of the plasmalemma rather than an "extraneous coat."     

A Bacterial Cell Wall Stain1

A new method is given to stain bacterial cell walls, especially of Escherichia coli and Bacillus cereus. The cells are smeared in water on a slide and, as soon as air-dry, are stained 3-4 minutes with a 1 % aqueous solution of new fuchsin. The smear is washed with water until the stain ceases to run and is then allowed to air dry. The slide is placed on a 50°C. warm plate for 10-20 seconds, and the smear is then covered with a thin film of a 1-2% solution of Congo red at a pH of about 9.5. The smear is ready for observation as soon as dry or it may be washed with water if desired before observation.     

Standard specimens for stain calibration: application to Romanowsky-Giemsa staining.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.     

Surface staining of sawed sections of undecalcified bone containing alloplastic implants.

A simple new method is described for the histological evaluation of bones containing alloplastic implants of ceramic and/or metallic materials. The undecalcified bone is embedded in acrylic resin and sectioned at 50-200 micrometer using a sawing microtome. One surface of the preparation is stained up to 10 micrometer deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is satisfactory for histological and morphometric evaluation.     

Selective light microscopic demonstration of the specific granulation of the rat atrial myocardium by lead-hematoxylin-tartrazine.

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

Selective Light Microscopic Demonstration of the Specific Granulation of the Rat Atrial Myocardium by Lead-Hematoxylin-Tartrazine

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Staining of Pathogenic Fungi with Gomori's Aldehyde-Fuchsin in Both Old and New Sections

Gomori's stain for elastic tissue can be applied directly to paraffin sections as a routine method for fungus capsules and membranes. Either freshly prepared, or old sections previously stained with hematoxylin-eosin, can be used. After applying the aldehydefuchsin stain, nuclei may be stained (or restained) with hematoxylin, and a general background stain obtained with either eosin or light green.     

Selective Light Microscopic Demonstration of the Specific Granulation of the Rat Atrial Myocardium by Lead-Hematoxylin-Tartrazine

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

Gallocyanin-Chrome Alum Counterstaining of Golgi-Kopsch Impregnations

A simple technique is described for counterstaining Golgi-Kopsch impregnations. The sections are first stabilized by the method of Geisert and Updyke and then stained in 0.15% gallocyanin and 5% chromium potassium sulfate for 45 minutes at 55-60 C. The sections are then rinsed, dehydrated to 70% ethanol, cleared in terpineol, mounted and coverslipped. This procedure results in a light to medium blue stain of those cells not impregnated by the silver chromate. The major advantages of this procedure over earlier methods are: (1) the counterstain does not fade and (2) since no differentiation is required, many sections may be stained simultaneously.