Brazilin-Toluidine Blue O and Hematoxylin-Darrow Red Methods for Brain and Spinal Cord

Tissue fixed in 10% formalin, formalin-95% ethanol 1:s CaCO₂ or phosphate buffer neutralized formalin, or methanol-chloroform 2:1, was dehydrated and embedded in paraffin or double-embedded by infiltration in 1% celloidin followed by a chloroform-paraffin sequence. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 24-26 C. For either method, mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. For brazilin-toluidne blue O, myelin was stained for 20-60 min, depending upon section thickness, in a self-differentiating solution consisting of: 0.15% Li₂CO₃ 75 ml; 6% brazilin in 95% ethanol, 25 ml; and NaIO₃ 75 mg. After a thorough washing, Nissl material was stained for 3-8 min in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; and 1% toluidine blue 0, 2.5 ml. For hematoxylin-Darrow red, myelin was stained for 2-6 hr in a self-differentiating solution consisting of: 0.15% Li₂,CO₃ 95 ml; 10% hematoxylin in 95% ethanol, 5 ml; and NaIO₃ 25 mg. After a thorough washing, Nissl material was stained for 20 min or less in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; Darrow red, 25 mg. This mixture was first boiled, cooled to room temperature and filtered. In both methods, washing, dehydration, clearing, and mounting completed the process. In the brazilin-toluidine blue technic, myelin sheaths were stained reddish purple; neuronal nuclei light blue with dark granules of chromatin; nucleoli dark blue; and cytoplasm blue with dark blue Nissl granules. In the hematoxylin-Darrow red procedure, myelin sheaths were blue-black; nuclei light red with dark granules of chromatin; nucleoli almost black; and cytoplasm red with bright red Nissl granules.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

The Selective Staining of Red Blood Cells

A method for the selective staining of red blood cells is described. Material is fixed in 10% neutral formalin in .85% NaCl and imbedded in paraffin or celloidin. Sections 6-10 μ are stained 1-5 minutes in chromotrope 2R. Basophilic and the less strongly acidophilic elements are decolorized with 5% phosphotungstic acid in 95% ethyl alcohol. Red blood cells and other strongly acidophilic elements that may be present in the preparation retain the chromotrope 2R. A counterstain of methyl blue may be used for staining the decolorized basophilic elements. As a result, erythrocytes are stained red by the chromotrope 2R, and basophilic elements blue, by the methyl blue. Less strongly acidophilic elements, having little affinity for either primary or secondary dye, are colorless or gray.     

Sudan Black and Osmic Acid as Staining Agents for Testicular Interstitial Cells

Two standard cytological techniques have heen modified to stain specifically the interstitial cells of the testis. In Method 1, the tissue is fixed in Zenker-formol or Regaud's fluid for several hours or overnight and subsequently postchromed in 3% K₂Cr₂O₇ for 72 hr at 37°C. After paraffin embedding, sections are cut at 5μ, dewaxed, brought down to 70% alcohol and stained in an unfiltered saturated solution of Sudan black in 70% alcohol for 10-30 min. Sections are washed briefly in 70% alcohol to remove all excess dye, differentiated, if necessary, in 50% alcohol, downgraded to water and mounted in Farrants' medium or glycerol jelly. Interstitial cells: deep blue black; remainder of testicular tissue: light blue. Method 2 is essentially the Champy-Kull technique but specific staining for mitochondria is omitted and the sections are downgraded to water; then they are mounted in Farrants' medium or glycerol jelly without further treatment. In this way osmicated lipoids are preserved. Interstitial cells: conspicuous due to the variable number of black granules in their cytoplasm; the remainder of the tissue: yellow.     

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.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Mast cells/eosinophilic granule cells of salmonids: staining properties and responses to noxious agents

Observations were made on mast cells/eosinophilic granule cells in swimbladder tissue spreads and sections of gills and intestinal tissues from species of the generaSalmoOncorhynchusSalvelinusCoregonusThymallus. Some individuals had been reared in captivity and others were caught in rivers or lakes, and both apparently healthy fish and fish with persistent inflammation, due to helminths or unknown causative agents, were included in the study. Acute responses to noxious agents were studied in swimbladder tissue spreads after intraperitoneal injections of inactivatedAeromonas salmonicida, compound 48/80 and hydrocortisone. The tissue spreads were fixed in ethanol and stained with thionin. Other tissues were fixed in a solution containing 4% formaldehyde and 5% acetic acid in methanol, and stained with May-Grünwald Giemsa combination dye, haematoxylin and eosin, or Alcian blue. Intestinal tissue histamine was assayed fluorometrically, and vascular responses to histamine and compound 48/80 were studied in perfused gill preparations. The staining properties of salmonid mast cells/eosinophilic granule cells resembled those of mammalian mucosal mast cells and globule leucocytes, with both acidophilic and basophilic components in their granules. May-Grünwald Giemsa staining revealed that in cells found in connective tissues the basophilic character was dominant, whereas the acidophilic character was most marked in those present in epithelia. The granule cells in swimbladder tissue spreads stained metachromatically with alcoholic thionin. Intraperitoneal injections of inactivatedA. salmonicidaproduced acute inflammatory reactions, with degranulation of mast cells/eosinophilic granule cells, in tissues of the swimbladder. Degranulation of the granule cells was also noticed after injection of compound 48/80. Massive degranulation of mast cells/eosinophilic granule cells in the swimbladder wall, followed by an acute inflammatory reaction, was induced by intraperitoneal injections of hydrocortisone. Persistent inflammation, e.g. in tissues infected with helminths, was accompanied by recruitment of mast cells/eosinophilic granule cells. Presence of many or few mast cells/eosinophilic granule cells in tissues of the intestine seemed to have no influence on the content of histamine, which was always low. Compound 48/80 produced increased resistance in the perfused branchial vascular bed, but effects of histamine were slight or completely absent. The responses of mast cells/eosinophilic granule cells of salmonids in acute and persistent inflammation, as revealed in the present investigation, are similar to the known responses of mammalian mast cells. Since their staining properties are also similar, the term ‘mast cell’ should be adequate.     

Bromphenol Blue as A Stain for Muscle Striations

Sections from formalin-fixed muscle are stained 4-24 hr with a 0.05% solution of bromphenol blue in 2% acetic acid, rinsed with water and placed in 0.5% acetic acid for 5-10 min. They are then treated 30 sec with tap water substitute (KHCO₃, 0.2 gm; MgSO₄, 2 gm; distilled water, 100 ml), rinsed, dehydrated in alcohol, cleared in xylene and covered in a polystyrene mountant Striatums of both cardiac and skeletal muscle fibers are fully resolved under oil immersion, against the blue background of the other parts of the fibers.     

Display of mitochondria in root tip cells

A method for displaying mitochondria and proplastids in root tip sections of Tradescantia paludosa and cereals was modified from Altmann and Volkonsky. Root tips were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.1, or acetate buffer, pH 4.8, for 3 hr, rinsed and postchromed overnight in 3% potassium dichromate, all at room temperature (20 C), dehydrated through a tertiary butanol series and embedded in ester wax. Four-micrometer sections were stained in hot acid fuchsin in aniline water, rinsed, treated with 1% sodium phosphomolybdate for 30 sec, rinsed and stained progressively with azure B for 3-10 min before being made permanent. Mitochondria and proplastids were stained brilliant crimson against a light blue cytoplasm with deep blue chromosomes. Previously reported difficulties with Altmann staining techniques are attributed to the erratic action of the classical fixatives used.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Modified Whipf's Polychrome: A Connective Tissue Stain with Special Application for Demonstrating Leishmania

A polychrome stain procedure was developed to demonstrate amastigotes of the protozoan parasite Leishmania braziliensis as well as cytoplasmic and other tissue components in cutaneous lesions of infected animals. The procedure is as follows: stain nuclei for 10 minutes with an iron hematoxylin containing 0.5% hematoxylin and 0.75% ferric ammonium sulfate dissolved in 1:1 0.6 N H₂SO₄:95% ethanol; rinse 4 minutes in distilled water. Cytoplasmic staining is achieved by exposing tissues for 10 minutes to a solution containing 0.25% Biebrich scarlet, 0.45% orange G, 0.5% phosphomolybdic acid and 0.5% phosphotungstic acid in 1% aqueous acetic acid. These first two solutions are modified from Whipf's polychrome stain. Sections are differentiated for 10 seconds in 50% ethanol, rinsed in water, stained 3 minutes in 0.1% aniline blue WS in saturated aqueous picric acid, rinsed in water and differentiated for 1 minute in absolute ethanol containing 0.05% acetic acid. Mordanting overnight in 6% picric acid in 95% ethanol produced optimal results.

This procedure was applied to sectioned material from experimental animals with various protozoa. Trypanosoma cruzi, Besnoitia Jellisoni, Toxoplasma gondii and especially Leishmania braziliensis were well demonstrated. Combining cytoplasmic dyes and phosphomolybdic-phosphotungstic acids into one solution afforded differential staining of tissues by Biebrich scarlet and orange G; connective tissues were stained by this solution. Substantially improved definition of connective tissues resulted after counterstaining. This procedure differs from the Massou sequence in which connective tissues are first stained by cytoplasmic dyes along with other tissues and then destained prior to specific counter-staining. in comparing dyes structurally related to Biebrich scarlet, it was found that Crocein scarlet MOO, but not Poncenu S, was an acceptable substitute. Sirius supra blue GL and Sirius red FSBA were not useful as replacements for aniline blue WS in this procedure.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Quad-Type Stain for the Simultaneous Demonstration of Intracellular and Extracellular Tissue Components

This technic for the simultaneous demonstration of several different tissue components works equally well on invertebrate and vertebrate tissue if they have been treated with nonchromate fixatives Sections 4-7 μ thick are stained 30 min in 1% Alcian blue, then treated with alkaline alcohol for 2 hr. They are stained in Verhoeff's hematoxylin for 4-6 hr, and rinsed in alcohol; stained in woodstain scarlet-acid fuchsin for 3 min, decolorized in 5% phosphotungstic acid for 20 min and finally stained 5-8 min in alcoholic saffron. Collagen and bone are stained yellow; elastin, myelin and nucleic acids, purple to black; muscle, chitin, cytoplasm, fibrinoid and acid secretion, bright red to lavender; ground substances and mucus, blue-green. Fibrous connective tissue, cartilage, bone and glandular epithelia are exceptionally well demonstrated by this method. Slides stained in this manner are well suited for color photomicrography and as demonstrations in the classroom.     

A Neoplastic Connective Tissue Mast Cell Capable of Continuous Growth In Tissue Culture

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.     

Effects of Ph on Post-Mortem Retention of Trypan Blue Vital Staining in Tissues of Mice

Vital staining of aortas from mice injected subcutaneously (daily for 5 days) with trypan blue was studied. In routine paraffin sections elastic membranes were observed to be well stained and other medial elements unstained following fixation in 10% formaldehyde (25% formalin) at pH 7-9. An identical pattern of vital staining was observed in specimens that had been immersed for 48 hr in saline solutions at pH 7-11. Elastic membranes were not stained, but intermembranous connective tissue was stained after the following: (1) fixation in 10% formaldehyde at pH 1-4 and in Lavdowsky's solution (ethanol, formaldehyde, water and glacial acetic acid), pH 2.3-2.8; and (2) immersion in saline for 48 hr at pH 14. Aortic elastic membranes were vitally stained after fixation by intracardiac perfusion with 10% formaldehyde (pH 7-8) but not after perhion with Lavdowsky's fixative (pH 2.3-2.8). Vital staining was limited to medial elastic membranes in sections of fresh aorta made in a cryostat or by a regular freezing microtome. The vital staining (coarse cytoplasmic granules of dye) within macrophages (Kupffer cells and others) and in cytoplasm of renal tubular epithelium was well demonstrated following use of all methods discussed above     

Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

A simple staining technique to demonstrate the sporozoite refractile globule in coccidian parasites

A staining technique was developed for locating the sporozoites of Eimeria spp. in both mammalian and avian hosts. Bouin-fixed tissues embedded in Paraffin wax and cut at a thickness of 3 micrometers were stained by a controlled sequence of Ehrlich's haematoxylin, acid fuchsin and aniline blue-orange G-acetic acid which took only 30 min to perform. The refractible globule of the sporozoite stained a brilliant crimson whilst the tissue had an overall blue colour, thus enabling rapid scanning of material under low-power magnification. The granules of eosinophils and heterophils also stained a brilliant crimson and their significance in immunological responses indicates a wider application for this technique.