Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Oxazine Dyes. Iv. Simultaneous Nuclear and Double-Contrast Cytoplasmic Staining from A Single Solution

A dye mixture, consisting of a celestine blue B dispersion (prepared according to Gray et al. 1956), orange G, and acid fuchsin in one solution, simultaneously stains nuclear elements and gives double contrast staining of cytoplasmic elements. Orange G, 0.16 gm, and acid fuchsin, 0.04 gm, dissolved in 100 ml of celestine blue B dispersion and adjusted to pH 0.8 gives, when applied for 1.5 min, results comparable or superior to other “triple contrast” stains on a wide variety of tissues. No differentiation other than that which occurs during dehydration is necessary.     

A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

A Quantitative Procedure for Estimating Cotton Fiber Growth

An improved procedure for quantitation of cotton fiber development, the “stain-destain” method, is reported. Toluidine blue 0 was used to selectively stain fibers subsequently destained in an acid-alcohol solution. Absorbance of the dyecontaining destaining solution was used as a measure of fiber development, and expressed in terms of total fiber units (TFU), one OD unit at 624 nm having been assigned the value of one TFU. Optimum conditions for the procedure, including staining and destaining times and solution to ovule ratios were determined: (1) 20 ovules with associated fibers stained for 15 sec in 80 ml 0.018% toluidine blue O, (2) nonabsorbed dye removed by 60 sec wash, (3) ovuls destajned in 100 ml glacial acetic acid-ethanol-water (10:95:5), (4) absorbance determined after one hr destaining. The procedure is deemed accurate and precise for the purpose intended—quadtation of fiber development as modified by phytohormones or other treatments. Data are shown correlating TFU with fiber length through 14 days postanthesis and an example is given in which the method was used to determine the effect of combined application of gibberellic acid and indoleacetic acid on in vitro cotton fiber development.     

Haematoxylin-Phloxine-Alcian Blue-Orange G Differential Staining of Prekeratin, Keratin and Mucin

This is a modification of Kreyberg's stain with Alcian blue 8GS used to stain acid much while phloxine B and orange G stain keratin and prekeratin. Procedure: Dewax formalin-fixed paraffin sections in xylene and hydrate through alcohol. Stain in Mayer's haemalum, 10 min; blue in tap water; wash in distilled water; stain in 1% phloxine, 3 min; wash in running water, 1 min; wash in distilled water; stain in 0.5% aqueous Alcian blue in 0.5 acetic acid, 5 min; wash in distilled water; stain in 0.5% orange G dissolved in 2.0% phosphotungstic acid, 13 min; dehydrate quickly in 2 changes of 95% alcohol and 2 changes of absolute alcohol; clear in several changes of xylene; mount in a synthetic resin. Acid mucopolysaccharides are stained turquois blue; prekeratin and keratin are orange to red orange.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Differential Cytophysiological Diagnosis of Cancerous and Normal Tissues

A method is offered for he differential diagnosis of cancer cells. It depends on the use of methylene blue decolorized with sodium thiosulfate (denoted here HLM, i.e. “hyposulfite methylene blue”); this is prepared by dissolving 800 mg. sodium thiosulfate in 10 ml. of 0.1% aqueous methylene blue and adding 3-5 drops of dilute (1:3) HCl. Frozen sections are treated with this reagent for 2-3 minutes, rinsed with a large amount of distilled water, then stained 2-3 minutes with 0.05% aqueous acid fuchsin. Staining should be performed in a darkened room. If all due precautions are observed, normal tissue appears blue, malignant tissue red.     

Improvements In The Paraffin Method

Dilute hydrofluoric acid alone and in conjunction with glycerin and ethyl alcohol was employed successfully to soften various types of refractory plant materials embedded in paraffin. Serial sections were cut at 6-8 μ and no appreciable deleterious effects on cell walls, cell contents, or staining procedures occurred. “Tannins” and “phlobaphene compounds” can be removed from tissues softened by this method by treating the sections for 12-48 hours with an aqueous solution of chromic acid, potassium bichromate and glacial acetic acid prepared according to the formula given by Johansen (1940).     

Further Experiments with the Masson Trichrome Modification of Mallory's Connective Tissue Stain

Several dyes, notably ponceau 2R, azofuchsin 3B, nitrazine yellow, and Biebrich scarlet may replace imported “ponceau de xylidin” in the Masson ponceau acid fuchsin mixture. Of these Biebrich scarlet appears to be the best and may be used without acid fuchsin.

A mixture of equal parts of 5% solutions of phosphomolybdic and phosphotungstic acids is much superior to either acid alone and gives adequate mordanting in 1 minute at 22°C.

With the fast green modification, times in plasma and fiber stains can be reduced to 2 minutes each. With anilin blue a 4-minute plasma stain is required. One-minute final differentiation in 1% acetic acid is adequate.

Primary mordanting of formalin material may be accomplished by 5 minutes in saturated aqueous mercuric chloride or 2 minutes in saturated alcoholic picric acid. Three minutes washing in running water is required after these mordants.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

A Method for Injecting Insect Tracheae Permanently

By the use of an alcohol insoluble dye (trypan blue), acetic acid, and a detergent (“Santomerse No. 3”), a resulting dye solution is obtained which will completely penetrate the tracheal system of an insect. The dye is injected by the use of a vacuum and by the pressure produced when the air is allowed to re-enter the dye vessel. The dye itself is permanently fixed in the tracheae by means of a fixing solution containing alcohol, acetic acid and barium chloride as its components. The material must be properly preserved after staining. It may be stored indefinitely in 70% alcohol, xylol, cedar oil or clove oil, depending on whether the material is to be used for sectioning or for whole mounts. Injected material may be sectioned in either celloidin or paraffin, or may be cleared and mounted in toto.     

Hot, Acidified Rhodamine B and Methylene Blue; A Differential Stain Dichromatic for Hair Follicular Keratins, Achromatic for Epidermal Keratin

Procedure:Cut paraffin sections and float on a 45-50 C water bath; spread silicone-rubber adhesive (Clear Seal-General Electric) thinly and evenly over 2/3 of the slide; pick up the sections from the floatation water with the coated slide; dry for 1.5 hr at 25 C and at 60 C for 0.5 hr; deparaffinize, and hydrate to water. Place 150 mg of rhodamine B and 150 mg of methylene blue each in separate 100 ml beakers and add 80 ml of 10% HCl to each beaker. Bring both solutions to a boil on a hot plate in a fume hood; immerse tissue sections in the boiling rhodamine B exactly 2 min; rinse in a beaker of 10% HCl 5 sec; immerse in the boiling methylene blue exactly 0.5 min; rinse in distilled water; blot dry; and mount in a silicone-rubber medium (Glass and Ceramic Adhesive—Dow Corning Corp.). Hair shaft keratin stains red; inner root sheath keratin and keratogenous zone of the hair shaft, blue green; epidermal keratin remains unstained. Pilomatrixornas show foci of both red and blue green keratin; epidermal and hair sheath (“sebaceous”) cysts remain unstained.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

A Simple Toluidine Blue—Basic Fuchsin Stain for Spermatozoa in Epoxy Sections

Differential staining of cell components of spermatozoa is readily accomplished in Epon or Araldite sections 0.5-1 μ thick from rat and hamster testis and epididymis, and stained as follows: 1% aqueous toluidine blue buffered at pH 6, 0.5-3 min at 90 C; washed in distilled water; 1% basic fuchsin in 50% alcohol, 3-5 min at 20-25 C; differentiated with 70% alcohol; allowed to dry; and mounted in a resin of high refraction (DPX was used). Results: acrosome, bright magenta; nucleus, deep blue; mitochondrial sheath of the middle-piece, pinkish purple; and tail, pale red. This procedure combined with staining of collagen by applying 2% aqueous phosphotungstic acid 1-2 min as a mordant, followed by 1% light green in 50% alcohol containing 1% acetic acid, 1-2 min at 20-25 C, gives polychromatic staining and is useful as a general stain for other epoxy-embedded tissues.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Antinociceptive activity of the volatile oils of Hyptis pectinata L. Poit. (Lamiaceae) genotypes

Hyptis pectinata L. Poit (Lamiaceae) is known popularly in Brazil as “sambacaita” or “canudinho” and is used in the treatment of inflammations, bacterial infections and ache. The antinociceptive activity of the volatile oils of six genotypes, at doses of 100, 200 and 400 mg/kg body wt., were investigated using abdominal writhe models induced by acetic acid and hot plate tests. The volatile oils of all the genotypes are composed mainly of sesquiterpenoids. All the genotypes showed antinociceptive effects in both models used; the SAM002 genotype showed the major inhibitory effect at dose of 100 mg/kg body wt. These results suggest that the volatile oil of H. pectinata has peripheral (writhe reduction) and central (time delay of thermal reaction) effects. These observations indicate that H. pectinata may be useful as an analgesic drug.