Curtis' Substitute for Van Gieson Stain

A triple staining method is described in which nuclear staining is by Weigert's hematoxylin. The cytoplasmic and collagen staining is effected by the Curtis substitute for Van Gieson, in which ponceau S is substituted for acid fuchsin. Nuclear staining is sharper than with Delafield's hematoxylin. The red of the collagen fibers is probably not subject to fading. Unlike Van Gieson, this method gives staining of reticular as well as collagen fibers. The advantages of the method are its simplicity and reliability. The use of this method is made possible by a new source of reliable samples of the ponceau S called for in this method.     

苏木精染色液的改进和使用

报道了一种新的特制苏木精染色液的配制、使用方法、染色效果及其使用价值。实验表明,该染色液全面而明显地优于通用的Harris染色液。     

Restoring and Lmmunohistological Examination of Feulgen Stained Avian Embryonic Tissues Using Iron Hematoxylin and Endothelial Cell Specific Antibody

The immunohistological method described here permits re-examination of previously Feulgen stained quail-chick chimera tissues for vascular development using the monoclonal antibody QH1 which specifically recognizes quail hemangioblastic cells. Weigert's iron hematoxylin has been used to restore faded or bleached Feulgen stained chimeric avian tissues. Species-specific differences in nuclear morphology are as evident with iron hematoxylin staining as they are with Feulgen staining.     

A rapid and simple method for visualizing milky spots in large fixed tissue samples of the human greater omentum

ABSTRACT

Milky spots are unique lymphoid structures in the greater omentum that participate in both immune homeostasis of the peritoneal cavity and formation of omental metastases. We developed a rapid and simple staining method to enable macro- or stereomicroscopic identification of these miniscule structures in large samples of fixed human greater omentum. By immersing approximately 6 × 4 cm samples of omental tissue in hematoxylin, these samples could be evaluated quickly for the presence of milky spots. We used an alum hematoxylin variant containing 1 g hematoxylin, 50 g aluminium ammonium sulfate, 0.2 g sodium iodide, 1 g citric acid and 50 g chloral hydrate. This staining method enabled us to determine the number, location, dimensions and topographical relation of milky spots to other structures. Our method also facilitates isolation of milky spots for further investigation. Hematoxylin imparts a blue color to the milky spots, which remains in place during further processing for paraffin embedding. This enabled easy recognition of milky spots during transfer through various solutions and permitted selection of relevant paraffin slides prior to additional staining.     

Application of immunohistochemical staining to detect antigen destruction as a measure of tissue damage

Electrocautery and directed energy devices (DEDs) such as lasers, which are used in surgery, result in tissue damage that cannot be readily detected by traditional histological methods, such as hematoxylin and eosin staining. Alternative staining methods, including 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to stain live tissue, have been reported. Despite providing superior detection of damaged tissue relative to the hematoxylin and eosin (H&E) method, the MTT method possesses a number of drawbacks, most notably that it must be carried out on live tissue samples. Herein, we report the development of a novel staining method, "antigen destruction immunohistochemistry" (ADI), which can be carried out on paraffin-embedded tissue. The ADI method takes advantage of epitope loss to define the area of tissue damage and provides many of the benefits of live tissue MTT staining without the drawbacks inherent to that method. In addition, the authors provide data to support the use of antibodies directed at a number of gene products for use in animal tissue for which there are no species-specific antibodies commercially available, as well as an example of a species-specific direct antibody. Data are provided that support the use of this method in many tissue models, as well as evidence that ADI is comparable to the live tissue MTT method.     

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.     

A Simple Method to Determine Hematoxylin Ripening

A simple spectroscopic method is proposed to control the “ripening” of Delafleld's hematoxylin solution during natural “ripening” or fast oxidation by different agents. The hematoxylin solution has ripened sufficiently for use with tissue sections staining if the absorption is 1.2-1.3 at 560 nm. The hematoxylin solution must first be diluted 15-fold using a 5% ammonium alum solution and the absorption must be measured in 0.5 cm cuvettes.     

On the Structure and Staining of Starch Grains of the Potato Tuber

Procedures are described for the differential staining of starch grains of the potato tuber with hematoxylin, and for double staining with safranin 0 and fast green FCF. The staining effects obtained are made possible by the action of a swelling agent. Staining with hematoxylin is preceded by the swelling action of formaldehyde. In staining with safranin 0 and fast green FCF, the formaldehyde is added to the staining solution. The results obtained are as follows: (1) a clavate-shaped, central structure composed of small particles arranged in definite layers is revealed within the grain; (2) differential staining of the locus of the grain and the lamellae alternating with it in a small region around the longitudinal axis of the grain; (3) the simultaneous staining and separation of the grain into a cone-shaped peripheral portion and a spherical body containing the locus of the grain which emerges from it; and (4) differential staining of a ring or layer of substance around a spherical refractive body within the grain.     

Delineation of human chromosome contour by heat treatment and hematoxylin staining

An unusual form of staining was observed as a result of heat treatment: chromosome contour became easily recognizable. Chromosome contour is optimally delineated by heat treatment of metaphase preparations in 0.06 M phosphate buffer, pH 6.8–7.0 at 95° C for 10 min, and subsequent staining with Heidenhain's hematoxylin. This result is obtained by a process similar to that in which Giemsa has been reported to stain particular foci considered to represent selective staining of constitutive heterochromatin. Mordanting with 3% ferrous ammonium sulfate produced maximum staining. However, hematoxylin (Harris) also stained the periphery of chromosomes. A transitional temperature existed between 70° C and 80° C in which contour became evident. When metaphase preparations were heated at 90–95° C, contour occurred on some chromosomes within 3 min and was demonstrated in 100% of the chromosomes analysed after 10 min of heat treatment.Supported in part by a grant to C. Romero-Sierra from the Defence Research Board of Canada (DRB 9350-24) and to E. A. MacKinnon from the Medical Research Council of Canada (MA-4135).     

Standardized Thionin-Eosin Y: A Quick Stain for Cytology

Two stains long used in exfoliative cytology, the hematoxylin-eosin Y and Papanicolaou stains, have not been standardized even today. Some dozens of hematoxylin and eosin and Papanicolaou staining recipes have been recommended in the literature. Consequently, the staining pattern of hematoxylin and eosin, and Papanicolaou stained cytological material varies from laboratory to laboratory. To a certain degree this is due to batch-to-batch variations of commercial samples of the natural dye hematoxylin (C.I. 75290). The present paper describes a simple, standardized and reproducible procedure using thionin bromide to replace hematoxylin in the hematoxylin and eosin stain.     

Staining of paraganglionic cells with lead hematoxylin

Lead hematoxylin staining in most paraganglionic cells is too faint to be observed in the light microscope (LM). Paraganglionic cells which appear stained (LM) are also known to contain more and larger specific granules (as seen in the electron microscope, EM) than those paraganglionic cells remaining unstained (LM). However, also specific granules in lead hematoxylin-negative paraganglionic cells (LM) bind lead ions (EM), which behaviour is the basis of lead hematoxylin staining. It is concluded that lead hematoxylin stains matrix material of specific granules but the amount of granule material mostly is too small to yield colour intensities to be observed in the LM.     

Use of eriochrome cyanine R for routine histology and histopathology: an improved dichromatic staining procedure

Abstract

A modified dichromatic iron-eriocyanine R (Fe-ECR) staining method is described. Staining obtained with this new technique generally was similar to that of hematoxylin and eosin (H & E). Cell nuclei were stained blue. Cardiac, smooth and skeletal muscle, and red blood cells, were stained different shades of red. Collagen fibers were stained different shades of orange, usually faintly. Decalcified bony tissue was stained pinkish violet. Epithelial cells were strongly stained deep shades of red, magenta and violet. Cartilage matrix, and goblet and mast cells were unstained. Although Fe-ECR staining differed too much from standard H & E staining to be a substitute for diagnostic purposes, the dichromatic method described might usefully replace van Gieson or trichrome stains, especially if muscle is of interest. A pH 0.95 staining solution was used to differentiate initially over-stained sections followed by washing in distilled water. This dichromatic technique is easier to perform and more precisely controllable than other ECR dichromatic methods. The entire procedure can be completed in less than 5 min. The technique has the advantages of greater technical simplicity and speed, a larger range of polychromasia, and a longer shelf-life than H & E. ECR also is more reliably available than hematoxylin and usually is less expensive.     

Mechanisms of chromation hematoxylin stains

Summary In chromation hematoxylin sequence stains of Weigert-Smith-Dietrich type an exploration is presented of the nature of chromium binding tissue end groups, of the valency of the bound chromium and of the mechanisms and conditions of its binding.At 2–4° C prolonged (2–8 weeks) mordanting in 2.5% K₂Cr₂O₇ at pH 3.5 engenders staining with acetic hematoxylin essentially limited to histochemical ethylenic sites, completely preventable by prior aqueous bromination and unaffected by sulfation or acetylation. Erythrocytes, myelin and bile casts are examples of reactive tissue elements. At 24° C and more so at 60° C there is increased reaction of muscle, cytoplasm, nuclei and other structures; the reaction is now partially blocked by acylations and is only partly prevented by bromination, indicating participation of hydroxyl groups. Deamination decreases reaction at 60° C of protein background, but not notably of myelin, erythrocytes, or bile casts. The previously reported carboxyl binding of hot chromation oxyphilia is almost inapparent when chromation is done at 3° C. Chromic acid is less selective in its action, producing some background staining even at 3° C; K₂CrO₄ engenders no acetic or neutral hematoxylin staining, even at 6.6% and 24 hour mordanting at 24° or 60° C.Chromium deposited from dichromate or chromic acid mordanting reacts with hematoxylin solutions at pH 2.5 to 7.0, that deposited from Cr III salts reacts only at pH 6–7. Mordanting with Fe III, Fe II, Cu II and Sn II salts yields hematoxylin staining respectively from pH 2.5, 4.0, 5.0 and 2.5 upward. After K₂Cr₂O₇ mordanting brief reductions and acid treatments restrict hematoxylin staining to the same neutral pH zone as that produced after Cr III mordanting, but the pH 7 staining capacity of Cr deposited from K₂Cr₂O₇ is more resistant to extraction agents than that from Cr III solutions. It is therefore concluded that the chromium deposited in dichromate mordanting is of a higher valency than Cr III and it is suggested that Cr IV is present and bound to double bond sites in ring esters in the same manner as has been formulated for Mn and Os in the attack of KMnO₄ and OsO₄ on ethylene double bonds.Supported by National Cancer Institute Grant No. C-4816, National Institutes of Health, Bethesda, Maryland. Presented in part at the Third International Congress for Histochemistry and Cytochemistry, New York 1968.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Method of total staining of monolayer cell cultures using vanadium hematoxylin]

Original method of cytological staining for monolayer cell cultures and outgrowth zone of tissue explants with vanadium hematoxylin is described. This staining method is simple, universal, reproducible and give opportunity to stain rapidly practically all cellular elements of cultivated monolayer with high degree of cytological resolution of intracellular structures (nucleus, cytoplasmic organelles etc.).     

An Azocarmine stain for Differential cell Analysis of the rat Anterior Hypophysis

A method of staining is described which is especially designed to facilitate differentiation of the cell types of the rat anterior hypophysis. Fixation in Zenker-formol solution is recommended. Pre-staining of the nuclei by a short immersion in alum hematoxylin is followed by mordanting in anilin alcohol and a 45 minute period of staining in azocarmine solution at 60d`C. The counterstains, acid green and orange G, are dissolved in clove oil to avoid destaining of the azocarmine.     

Hematoxylin as a Pituitary Stain

The accurate picture of the acidophil granules of the anterior pituitary which is provided by iron hematoxylin can be combined with the differential staining of the basophils by either the periodic acid-Schiff (PAS) or combined aldehyde-fuchsin-PAS procedures. To accomplish this the two stages of the iron hematoxylin technique are separated so that mordanting in iron alum precedes and application of hematoxylin follows the basophil procedures.     

An Evaluation and Modification of Cole's Hematoxylin

Consistency in staining with an alum hematoxylin is possible by the routine use of fresh staining solutions. A modification of Cole's hematoxylin is so easily prepared that fresh staining solutions present no problem. The staining solution consists of 100 ml 1.2% aqueous KA1(SO₄)₂ .12 H₂O, 1 ml 10% alcoholic hematoxylin and 2 ml 1% iodine. Mix, place in paraffin oven overnight and stain sections 5 minutes. The three solutions can be kept as stock solutions for years.     

News from the Biological Stain Commission

Abstract

The origins of repeated hematoxylin shortages are outlined. Lack of integration in the hematoxylin trade exacerbates the problems inherent in using a natural product. Separate corporations are engaged in tree growth and harvesting, dye extraction, processing of extracts to yield hematoxylin, and formulation and sale of hematoxylin staining solutions to the end users in biomedical laboratories. Hematoxylin has many uses in biological staining and no single dye can replace it for all applications. Probably, the most satisfactory substitutes for aluminum-hematoxylin (hemalum) are the ferric complexes of celestine blue (CI 51050; mordant blue 14) and eriochrome cyanine R (CI 43820; mordant blue 3, also known as chromoxane cyanine R and solochrome cyanine R). The iron-celestine blue complex is a cationic dye that binds to nucleic acids and other polyanions, such as those of cartilage matrix and mast cell granules. Complexes of iron with eriochrome cyanine R are anionic and give selective nuclear staining similar to that obtained with acidic hemalum solutions. Iron complexes of gallein (CI 45445; mordant violet 25), a hydroxyxanthene dye, can replace iron-hematoxylin in formulations for staining nuclei, myelin, and protozoa.     

Notes on Technic: Mahon's Myelin Stain for Celloidin-Embedded Nervous Tissue

Two methods commonly used to stain myelin sheaths are Kluver and Barrera's luxol fast blue (Kluver and Barrera 1953) and Weil's iron hematoxylin (Weil 1928). Both require differentiation of the stain; in addition, the Kluver-Barrera method specifies 16-24 hour staining. A third method for the selective staining of myelinated axons is that of Mahon (1938), which was introduced for use with paraffin-embedded autopsy tissue. The procedure possesses two distinct advantages since it requires: (1) no differentiation of the stain and (2) only 1 hour staining. Loyez's (1910) myelin stain for celloidin embedded tissue is similar to Mahon's but calls for long staining followed by differentiation. This report describes the application of Mahon's method to celloidin-embedded experimental tissue and emphasizes its utility for staining tissues to be used for reconstructing microelectrode penetrations (fig. 1) and for demonstrating the effect of experimental lesions (fig. 2).