Chlorantine fast green BLL as a stain for callose in oak phloem

Sections of oak bark were stained with chlorantine fast green BLL, used as a 0.25% aqueous solution. All tissues were unstained, except for local deposits of material associated with phloem cell walls, which stained deep green. This green-staining material also stained specifically with resorcinol blue and with the aniline blue fluorescence technique, the usual histochemical tests for callose. The chlorantine fast green-staining material was removed from sections by treatment with a beta-1,3-glucan hydrolase. It is concluded that chlorantine fast green BLL stains callose in plant sections and is a useful additional stain for the histochemical detection of this polymer.     

Alcian Blue Staining of Nuclear Chromatin in Insect Gut After Beta-Glucuronidase Treatment

Sections of the gut (ventriculus and proventriculus) of the cockroaches, Blattus germanicus and Blaberus giganticus, were prepared after fixation in Carnoy's solution. In sections treated with beta-glucuronidase and hyaluronidase (1 nig per 1 ml at pH 7.0), the nuclear chromatin of the epithelium stained deeply with alcian blue (0.1% in 2% acetic acid). The sites of this staining coincided with the green-staining components seen in untreated control sections stained by methyl green-pyronin. Moreover, the alcian blue staining after this treatment agreed closely with the sites of positive Feulgen reaction in control sections. Prior treatment in deoxyribonuclease (1 mg per 10 ml of glass-distilled water) before digestion in beta-glucuronidase nullified the alcian blue staining of the chromatin. Ribonuclease had a similar effect except that after its action the chromatin would still stain with nuclear fast red.     

In Vivo Staining by Dis and Trisazo Dyes, Especially of Bone and Elastic Tissue

The localization and retention of dis and trisazo dyes in connective tissues and bone was studied in rats and rabbits. Chlorazol fast pink, 5% in 0.9% NaCl. was injected intraperitoneally, 25 mg/kg each day for 2 days in newborn, growing, mature and 17-18 day pregnant rats, and up to 5 days in young rabbits. The dye was also injected at different time intervals during the development of strontium-induced rickets in growing rats, and in animals with abscess walls following subcutaneous injection of 0.1 ml turpentine. Animals were killed at-intervals thereafter, and comparison of in vivo staining of 5% solutions of chlorazol fast pink, chlorantine fast red, chlorazol black E, chlorazol sky blue, chlorazol sky pink, chlorazol green, chlorazol violet, pontamine green and pontamine sky blue was made by intraperitoneal injection in rats. Soft and hard tissue specimens were embedded in polyester resin or in paraffin wax and sectioned at 5-7 μ. Chlorazol fast pink stained some connective tissues and growing bones. The main intensity of staining occurred within 24 hr and gradually decreased but was still detectable after 6 mo in elastic tissues. In thin plastic sections, colouration was brilliant, not in osteoid tissue, but at calcifying bone margins and in elastic fibres. Dye localized at calcifying bone margins was incorporated within calcified tissues and then subsequently lost through remodelling. Such staining was not seen in paraffin-embedded material. Dye uptake was greatly reduced in rachitic rats, and wide osteoid seams were coloured faint pink, but where calcification was still occurring, colouration was brilliant. Similarly collagenous tissue in abscess walls was only lightly stained, in contrast to brilliant colouration of elastic tissues and macrophages. Of the 9 dyes tested, only chlorazol fast pink and chlorazol sky blue stained bone and elastic tissue in vivo. This prolonged retention and staining by these 2 dyes, unlike the others, was associated with their presence in the proximal convoluted tubules of the kidney.     

A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

A new fluorescent staining method for callose of microspore mother cells during meiosis

To observe the dynamic behavior of callose of microspore mother cells during meiosis, we developed a convenient, rapid and efficient staining method using an improved carbol fuchsin/aniline blue solution. The stained microspore mother cells during meiosis showed yellowish green callose, red cytoplasm and dark red chromosomes when excited with blue light, which produced a contrasting image with a three-dimensional effect. When stained with only improved carbol fuchsin solution, the cells had red cytoplasm and chromosomes when excited with green light. The improved carbol fuchsin solution can be used to replace other more expensive DNA-specific dyes, such as DAPI and H33258, to reduce experimental costs.     

Combined Bright Field and Fluorescence Staining of Paraffin Sections for Studying the Fertilization Process in Plants

PAS-toluidine blue O—aniline blue staining of paraffin sections allows study of histological and cytological detail while retaining aniline blue induced fluorescence in all “callose sites”. Because most autofluorescence is eliminated by the PAS-toluidine blue prestaining, the detail and contrast of the fluorescence image is superior to slides stained in aniline blue alone. Slides are stained by the PAS reaction, 0.03% toluidine blue O, alkaline 0.005% aniline blue, and mounted directly in aqueous mounting medium.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Luxol Fast Blue as a Selective Stain for Alpha Cells in the Human Pituitary

Human pituitaries fixed in Bouin's fluid or 10% formalin were stained by the PAS, Masson trichrome and luxol fast blue methods. By comparing adjacent sections stained by these 3 methods it was found that the alpha cells which are PAS negative, but stained red by the Masson trichrome method, were intensely stained by luxol fast blue. The beta cells which are stained blue by the PAS and Masson methods were not stained by luxol fast blue. Similar observations were made on a series of pituitaries from 8 other mammalian species. It is concluded that luxol fast blue is a selective stain for alpha cells in the mammalian pituitary.     

Staining Paraffin Embedded Sections of Scald of Barley before Paraffin Removal

Staining of paraffin embedded sections with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differentiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalis. Crystal violet, toluidine blue O and aniline blue also successfully stained fungal structures of R. secalis in barley leaf tissues. Staining of embedded sections before paraffin removal allows simple processing of a series of sections, saves time and reduces solvent consumption.     

Examining the microanatomy of various fetal body parts using the orcein-picroindigocarmine staining method

It is difficult to distinguish muscle, vessel, nerve, fascia, tendon and many other structures in fetal sections. The aim of the study reported here was to research the practicability of orcein-picroindigocarmine staining for distinguishing anatomic structures in histological sections containing complex structures. Histological serial sections of hand, foot, head and neck of four 18- to 20-week old fetuses were used. Bone matrix was stained bright blue, collagen fibers tones of green to blue, elastic fibers brown, cartilage matrix tones of pink, and chondroblasts blue. Muscles and erythrocytes were stained yellow to green, epidermis reddish brown, skin appendices including hair and nail stained light yellow to green.     

Examining the microanatomy of various fetal body parts using the orcein-picroindigocarmine staining method

It is difficult to distinguish muscle, vessel, nerve, fascia, tendon and many other structures in fetal sections. The aim of the study reported here was to research the practicability of orcein-picroindigocarmine staining for distinguishing anatomic structures in histological sections containing complex structures. Histological serial sections of hand, foot, head and neck of four 18- to 20-week old fetuses were used. Bone matrix was stained bright blue, collagen fibers tones of green to blue, elastic fibers brown, cartilage matrix tones of pink, and chondroblasts blue. Muscles and erythrocytes were stained yellow to green, epidermis reddish brown, skin appendices including hair and nail stained light yellow to green.     

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.     

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.     

A Rapid Safranin-Metal Phthalocyanine Double Staining Technique for Plants

Pure metal 4.4',4',4'-tetxa-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4',4'-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4',4'-tetra-sulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log ε = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.     

Mucopolysaccharides in the dog spinal cord and dorsal root ganglia. A histochemical study.

A histochemical study of mucopolysaccharides in the dog spinal cord and dorsal root ganglia is reported in this paper. The histochemical techniques used were the following: PAS, colloidal iron, toluidine blue (pH 5.4 and 3.5), thionine (pH 5.4 and 3.5) and alcian blue 8GX (pH 1 and 2.5). Some histological stains were used also. Two types of neurons could be observed in spinal cord sections stained with colloidal iron techniques. In some neuron a border line of mucosubstances could be seen. In the dorsal root ganglia, different patterns of Nissl bodies distribution in neurons were described. This different distribution of Nissl bodies is associated with different metachromatic colorations of neurons. By using the colloidal iron method, two types of neurons were also revealed in the dorsal root ganglia: some neurons are of a yellow, small-sized and star-shaped type and others are of a light green, larger and round-shaped type. Mucosubstances in the endoneurium and perineurium of nerve fibers, in the Ranvier nodules and in the Schmidt-Lantermann incisures were observed. The possibility that the functional rhythm in some cases might be responsible for the difference in coloration between the dorsal root ganglia neurons is suggested.     

从甲状腺肥大细胞探讨胎儿器官肥大细胞的差异

研究肥大细胞在人胎儿甲状腺发育中数量、分布及组化性质的改变,以探讨胎儿器官发育中肥大细胞的差异。取45例不同胎龄的人胎甲状腺石蜡切片做甲苯胺蓝染色和阿尔辛蓝－－藏红染色,并测定肥大细胞的临界电解质浓度值及进行硫酸小蘖硷荧光染色。结果显示：3月龄胎儿甲状腺内开始出现肥大细胞,数量极少,主要分布在被膜及小叶间结缔组织内,甲苯胺蓝染色肥大细胞颗粒呈淡紫蓝色,阿尔辛蓝－－藏红染色呈蓝色,临界电解质浓度值较低,硫酸小蘖硷染色未见显黄色荧乐的肥大细胞,从3月龄到足月随着胎龄增长,肥大细胞数量缓慢增多,8月龄时肥大细胞经甲苯胺蓝染色,其颗粒呈紫红色,阿尔辛蓝－－藏红染色出现少量含红色和红蓝混合染色颗粒的肥大细胞,临界电解质浓度值偏高,可见少量显黄色荧光的肥大细胞,结果表明：在人胎儿3月龄时甲状腺发育中开始出现肥大细胞,但随胎儿发育肥大细胞的组化性质改变不明显。     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.     

Association of polyphosphate with protein in freeze-substituted sclerotia ofSclerotinia minor

Summary In freeze-substituted sclerotia stained with aqueous toluidine blue O, metachromatic material was found throughout the cytoplasm in discrete granules. It was also distributed evenly throughout spherical and elongate protein bodies. This material stained at low pH and was extracted by cold acid, indicating that it was polyphosphate. Retention of metachromatic material was much greater than previously reported in chemically fixed, conventionally processed sclerotia. X-ray microanalysis of dry-cut, unstained sections of freeze-substituted sclerotia confirmed that phosphorus was distributed evenly throughout the protein bodies and was not localised in discrete granules but phosphorus levels in the cytoplasm were very low. It is concluded that polyphosphate is lost during conventional preparation procedures but retained in dry-cut, unstained sections of freeze-substituted material. However, when freeze-substituted sections were stained with toluidine blue O, water soluble polyphosphate was extracted and subsequently precipitated in the cytoplasm as polyphosphate granules. Therefore it is considered that polyphosphate granules are an artefact, and that protein bodies are the major site for storage of phosphorus in this fungus.Abbreviations STEM scanning transmission electron microscope - ER endoplasmic reticulum