Showing papers in "Biotechnic & Histochemistry in 1954"
TL;DR: Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr, and subsequently treated with 0.05% potassium permanganate for 4–10 min, and covered in neutral synthetic resin.
Abstract: Frozen sections of formalin-fixed brains containing surgical lesions, were treated with 15% ethanol for 0.5 hr., soaked in 0.5% phosphomolybdic acid for 0.25–1.0 hr., and subsequently treated with 0.05% potassium permanganate for 4–10 min. (The duration of the latter treatment is critical and individually variable). Subsequent procedure is as follows: decolorize in a mixture of equal parts of 1% hydroquinone and 1% oxalic acid; wash thoroughly and soak sections in 1.5% silver nitrate for 20–30 min.; ammoniacal silver nitrate (silver nitrate 0.9 g., distilled water 20 ml., pure ethanol 10 ml., strong ammonia 1.8 ml., 2.5% sodium hydroxide 1.5 ml.) 0.5–1.0 min.; reduce in acidified formalin (distilled water 400 ml., pure ethanol 45 ml., 1% citric acid 13.5 ml., 10% formalin 13.5 ml.) 1 min.; wash, and pass section through 1% sodium thiosulfate (0.5–1.0 min.); wash thoroughly and pass sections through graded alcohols and xylene (3 changes); cover in neutral synthetic resin.
810 citations
TL;DR: Comparison with sections treated with periodic-acid-Schiff (PAS) showed that some types of reticulin fibers are PAS-positive and others PAs-negative, and that in both categories there are Alcian-blue- positive and Alcian -blue-negative fibers.
Abstract: Paraffin sections of fixed material are (1) stained with Ehrlich's hemalum as usual, (2) stained in a 0.5% Alcian blue 8 G solution in 0.5% acetic acid for 30 minutes, (3) after rinsing, treated with 1% phosphomolybdic acid for 10 minutes, (4) after rinsing, stained in 0.5% chlorantine fast red 5 B for 10–15 minutes, (5) after rinsing, mounted in balsam or synthetic resin in the usual way. Mucin, granules of the mast cells, cartilage matrix and some types of reticulin fibers are stained blue-green; collagen fibers and ossein are red. Alcian blue stains selectively the structures containing mucopolysaccharides. Comparison with sections treated with periodic-acid-Schiff (PAS) showed (1) that some types of reticulin fibers are PAS-positive and others PAS-negative, (2) that in both categories there are Alcian-blue-positive and Alcian-blue-negative fibers. In cartilage matrix, Alcian blue gives pictures different from those obtained with PAS. The use of the combined Alcian blue, chlorantine red technic is reco...
197 citations
TL;DR: An Aceto-Carmine Glycerol Jelly for Use in Pollen-Fertility Counts was proposed by as mentioned in this paper, who used it for counting the number of fertility counts.
Abstract: (1954). An Aceto-Carmine Glycerol Jelly for Use in Pollen-Fertility Counts. Stain Technology: Vol. 29, No. 5, pp. 277-277.
169 citations
TL;DR: The hydrochloric-acid-Schiif reaction has been valuable since Feulgen reported it in 1924 and appears to be as practical as hydrolysis in 1 N HCl at 60° for 4–6 minutes.
Abstract: Among the cytochemical methods for demonstrating desoxyribonucleic acid, the hydrochloric-acid-Schiif reaction has been valuable since Feulgen reported it in 1924. A difficulty in that technic is that the section may come loose from the slide; this is caused by hydrolysis at 60° C. When sections were hydrolyzed by 1, 3, 5 or 6 N HCl at room temperature for 15 minutes, adequate hydrolysis and the strong development of color occurred with 5 N HCl. Similarly successful results were obtained with 5 N nitric acid hydrolysis for 10 minutes. Both procedures appear to be as practical as hydrolysis in 1 N HCl at 60° for 4–6 minutes.
80 citations
TL;DR: Root tips were soaked 1–2 hours at 18–20° C. in a freshly prepared saturated aqueous solution of α-bromonaphthalene, fixed in a mixture consisting of 1% chromic acid, 5 ml.
Abstract: Root tips were soaked 1–2 hours at 18–20° C. in a freshly prepared saturated aqueous solution of α-bromonaphthalene, 1–2 hours in water, and fixed in a mixture consisting of 1% chromic acid, 5 ml.; 2% osmic acid, 1 ml.; and 0.002 M 8-oxyquinoline, 1 ml. for 0.5–1.0 hour at 10–14°C. They were then successively treated as follows: water 1–2 minutes; 1% H2SO4, 10–15 minutes; water, 1–2 minutes; 1% CrO3, 0.5–1.0 hour; water 3–5 minutes; 1 N HCl, 45 minutes at 60°C; water 3–5 minutes; leucobasic fuchsin (de Tomasi) 0.5–1.0 hour. The brightly stained tips of the roots were cut off and squashed in the usual manner in a drop of aceto-carmine under a cover glass. The cover glass was sealed with paraffin and the slide stored overnight. The paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. Final processing consisted of passing the slide through pure n-butyl alcohol; a 1:1 mixture of redistilled turpentine and n-butyl alcohol (to remove-the osmic acid stain); n-bu...
51 citations
TL;DR: Frozen sections, 15–50 µ thick, are soaked for 5 minutes or longer in a mixture of equal parts of 1.5% aqueous gelatin and 80% alcohol, and teased onto a slide, anchoring the section to the slide.
Abstract: Frozen sections, 15–50 µ thick, are soaked for 5 minutes or longer in a mixture of equal parts of 1.5% aqueous gelatin and 80% alcohol, and teased onto a slide. After allowing excess fluid to evaporate, sections will be moist and can be blotted with filter paper that may require dampening with 95% alcohol. Immersed in 95% alcohol, the remaining gelatin will congeal, anchoring the section to the slide. If necessary, the sections can subsequently be coated with celloidin.
44 citations
TL;DR: A method is described for embedding and sectioning hard, undecalcified bone, which is designed for use by technical personnel and the results obtained are illustrated.
Abstract: A method is described for embedding and sectioning hard, undecalcified bone, which is designed for use by technical personnel. Bone fixed in a variety of ways is progressed through alcohols to ether-alcohol and then infiltrated with ether-alcohol solvented plastic (plasticized nitrocellulose) by a combination of centrifugation and high pressure embedding technics. The ether-alcohol is evaporated in a partially closed container in a manner similar to that employed in celloidin embedding, but differs from the latter by the removal of all of the solvent. Celloidin is the source of nitrocellulose and Amoil-S, the added plasticizer. Undecalcified adult bone of all types is readily cut at a thickness of 5–8μ on a heavy duty sliding microtome. The sections are then mounted on gelatinized slides. The procedures for preparing strip film radio-autograms of bone sections and subsequent staining of the preparation are given. The results obtained are illustrated.
40 citations
TL;DR: Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites, and show that while the present Protargol S is usable for protzoa, it is still inferior to the old German variety.
Abstract: Seven samples of silver protein (Protargol type) were tested on flagellate protozoa from the alimentary tract of frogs, golden hamsters, and termites. The samples consisted of one of Protargol (Winthrop's prewar German), one of Protargol S (Winthrop-Stearns, Inc., present manufacture, Commission Certified), and five of the experimental batches (4Z, 20, 20B, 22, and 36) by H. A. Davenport and collaborators (1952). The pre-war Protargol is rated best and batch 22 second best for staining of all flagellates. Protargol S gave uniform, but only fair results, with all organisms, while batch 20B, better than Protargol S in several instances, was poorer in a few. Thus Protargol S and batch 20B are rated third, as about equal. Batch 4Z is rated fourth; and batch 20, which stained some species, fifth. Batch 36 stained no protozoa. The tests show that while the present Protargol S is usable for protozoa, it is still inferior to the old German variety. Since some of the experimental batches, none of which was made by...
39 citations
TL;DR: Slices of cat brains that had been fixed in 10% aqueous formalin for various periods from 2 days to over a year were subjected to different types of chromation prior to impregnation with silver nitrate, finding acetic acid was somewhat preferable to formic as the acidifying agent.
Abstract: Slices of cat brains that had been fixed in 10% aqueous formalin for various periods from 2 days to over a year were subjected to different types of chromation prior to impregnation with silver nitrate. Acid solutions of Al, Ba, Ca, Cd, Ce, Co, Cu, Fe, K, Ni, Sr and Zn chromates were tested for usefulness as chromating agents. The chromates of Cd, Co, K, Sr and Zn were found to be best; Ca, Ce and Ni gave positive results, but Al, Ba, Cu and Fe were quite unsatisfactory. Acetic acid was somewhat preferable to formic as the acidifying agent. A formula consisting of potassium chromate, 5% aq., 100 ml. and glacial acetic acid 6–8 ml. was found to be similar in action to comparable mixtures that contained the chromate of Cd, Co, Sr or Zn. Brain slices chromated 24–48 hours in these acidified chromates and silvered in 0.75–1.0% silver nitrate for 12–24 hours at 37–40° C. gave at least three times as many good preparations as similar specimens chromated with plain potassium dichromate solution.
29 citations
TL;DR: A semipermanent slide containing a concentration of well-spread, randomized cells suitable for rapid mitotic frequency determination is obtained, and scores about 1,000 cells as to nuclear stage are given.
Abstract: Treating a Feulgen stained (10 min. acetic-alcohol fixation, 8 min. hydrolysis) onion root tip with 5% aqueous pectinase for 6 hours causes it to disintegrate on shaking in 1 ml. water into a suspension of stained cells, either individual or in small groups. Vicia faba and pea root tips require 15 minutes hydrolysis and 12 hours pectinase treatment. Absence of cell destruction allows absolute cell-number determination by Brown and Rickless' counting chamber technic. By centrif uging the cells down, resuspending them in 2 drops of a Karo syrup-phosphate buffer mixture (2 parts Karo to 1 part 0.5M, pH7 buffer) and mounting a small drop of the now concentrated suspension, a semipermanent slide containing a concentration of well-spread, randomized cells suitable for rapid mitotic frequency determination is obtained. Scoring about 1,000 cells as to nuclear stage gives a representative, workable statistical sample and takes only 20–30 minutes if interphases are recorded on a mechanical tally. Permanent slides c...
25 citations
TL;DR: Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope, and nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy.
Abstract: Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast ...
TL;DR: To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges, and experiments showed that theFeulgen reaction was optimum at pH 3, the Mc manus reaction at pH 2.4.
Abstract: Schiff reagents were made by two methods. The first procedure gave a Schiff reagent of pH 1.8–2.4. It was accomplished by passing sulfur dioxide into 0.5% aqueous fuchsin solution at room temperature, stopping at reddish violet, and decolorizing allowed to occur on standing. In another method, 1.5 ml. of 5.6% sulfurous acid was added to 100 ml. 0.5% fuchsin solution and the mixture produced in several hours a colorless Schiff reagent of pH 3. The solution remained unchanged for some weeks when kept stoppered in a refrigerator.To test these Schiff reagents, histochemical examinations were carried out with Feulgen and McManus reaction in various pH ranges. These experiments showed that the Feulgen reaction was optimum at pH 3, the McManus reaction at pH 2.4.
TL;DR: Apothecia of Pyronema confluens Tul. were stained by the Feulgen reaction and then squashed and mounted in propiono-carmine.
Abstract: Apothecia of Pyronema confluens Tul. were stained by the Feulgen reaction and then squashed and mounted in propiono-carmine. Exceptionally clear differentiation of the chromosomes was obtained by this method. Snail stomach cytase was used as an aid in flattening the asci and a simple method for its extraction is described.
TL;DR: A paraffin ribbon is fastened on a perforated metal plate having a rectangular hole wide enough to allow the ribbon to be suspended freely and pressed against the albuminized photographic emulsion.
Abstract: A paraffin ribbon is fastened on a perforated metal plate having a rectangular hole wide enough to allow the ribbon to be suspended freely. The desired section is centered over the hole and straightened by softening the paraffin over a hot plate and blowing gently on the section. The section is then cut loose on the end of a rubber stopper under a safelight and pressed against the albuminized photographic emulsion. Mayer's albumin does not affect the sensitivity or produce chemical fog in the emulsion.
TL;DR: Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3–6 mm slices and impregnated by Golgi's dichromate-silver procedure, and counter-staining was most successful when applied to freshly cut sections.
Abstract: Brains of cats that had been fixed 2 months or longer in 10% formalin were cut into 3–6 mm. slices and impregnated by Golgi's dichromate-silver procedure (6% dichromate solution, 4–6 days; 1.5% silver nitrate solution 2 days). Sections 100 µ thick were cut after embedding in low melting point paraffin. Three changes of xylene and three of absolute alcohol were followed by staining 3–5 minutes in a saturated solution of thionin in absolute alcohol. The sections were dipped quickly in absolute alcohol and cleared in xylene, then differentiation was effected by an equal-parts mixture of absolute alcohol and xylene. A final clearing in three changes of xylene and mounting in Permount completed the process. Counter-staining was most successful when applied to freshly cut sections.
TL;DR: The control of the endpoint of decalcification of histological specimens can be done by X-ray fluoroscopy by mounted under the table in the laboratory using a leaden tube and closely adapted to a hole in the table.
Abstract: AbstactThe control of the endpoint of decalcification of histological specimens can be done by X-ray fluoroscopy. An X-ray machine is mounted under the table in the laboratory. A leaden tube leads from the X-ray machine and is closely adapted to a hole in the table. The opening on the table surface is covered with a 5 mm. thick plastic plate on which is placed the object to be examined. The object is observed in a regular fluoroscope. All precautions to prevent direct and indirect X-ray irradiation of the observer should be taken.
TL;DR: Numerous mitotic plates of contracted and well-spread chromosomes may be obtained from root tips of plants set on melting ice or snow overnight at room temperature by mordanted in a mixture of alcohol plus 21/2 parts of 3% ferric ammonium sulfate for 3 hours to overnight.
Abstract: Numerous mitotic plates of contracted and well-spread chromosomes may be obtained from root tips of plants set on melting ice or snow overnight at room temperature. After 1:3 acetic-alcohol fixation for 0.5 to 3 hours the material is mordanted in a mixture of 7 parts of alcohol plus 21/2 parts of 3% ferric ammonium sulfate for 3 hours to overnight. This solution may be used as storage fluid for flower buds. Deep chromosome coloration without precipitates is secured by staining in a few drops of aceto-carmine for 10–15 minutes after which the tissues are softened by heating in aceto-carmine diluted with 3 parts of 45% acetic acid.
TL;DR: Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems.
Abstract: Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.
TL;DR: If sections of ground bone are first coated with a plastic solution before applying the usual mounting medium and a cover glass, excellent optical differentiation results.
Abstract: If sections of ground bone are first coated with a plastic solution before applying the usual mounting medium and a cover glass, excellent optical differentiation results. The solution consists of 28 g. of Parlodion dissolved in 250 ml. of either butyl or amyl acetate. Bone sections are prepared by grinding to the desired thinness, dehydrated with alcohol, air dried, dipped in the solution, freed of surface bubbles by agitation, and placed on a slide. After allowing the preparation to dry thoroughly, the customary mounting medium and cover glass are applied. The plastic seal prevents the escape of air from the lacuni and canaliculi, preserves the natural differentiation of bone tissue, and also permits it to be viewed with a polarizing microscope.
TL;DR: The formula proposed by Swank and Davenport (1935) was modified and applied to human and macaque nervous material and enhancement of staining of the degenerating fibers occurred and the different structures of the normal tissue were more easily identified.
Abstract: The formula proposed by Swank and Davenport (1935) was modified and applied to human and macaque nervous material. Three groups of experiments were performed and the following observations were made. (1) Diluting the osmic acid component, without altering the relative concentration of the other constituents of the solution resulted in practically no staining of the degenerated fibers. (2) When all constituents of the staining solution were used in much lower concentration than previously suggested, enhancement of staining of the degenerating fibers occurred and the different structures of the normal tissue were more easily identified. (3) At low concentrations of osmic acid and potassium chlorate, the contrast was diminished and artifacts produced by increasing the concentration of acetic acid or formalin or both. The new formula, based on the present results, consists of osmic acid, 0.5%, 11 ml.; potassium chlorate, 1%, 16 ml.; formalin (cone), 3 ml.; acetic acid, 10%, 3 ml.; and distilled water to make ...
TL;DR: There was greater difference between types of tissue than between species so that regardless of species, leaf tissue was easiest and root tips the most difficult to freeze and dry.
Abstract: The freeze-dry method of preparing tissue for histological observation has been applied to a large variety of plant material with good results. This method involves freezing the tissue rapidly and dehydrating at a temperature below −30°C. under vacuum with a desiccant. The tissue, when dry, is infiltrated with paraffin under vacuum. The apparatus uses a liquid nitrogen-cooled condenser as the desiccant and the tissue is infiltrated with paraffin while in the original vacuum. Using root, stem, leaf, and reproductive tissue of common experimental species such as Vicia faba, Zea mays, Allium cepa, Lillium longiflorium, Pisum sativum and Phaseolus vulgaris, the effect of thickness on drying time was studied. It was found that there was greater difference between types of tissue than between species so that regardless of species, leaf tissue was easiest and root tips the most difficult to freeze and dry. Leaf segments required 1–2 days to dry while root material required 6 days or more. In all tissue except le...
TL;DR: A new type of apparatus for cutting 100–200µ sections of bone is described, consisting of an especially designed bone vise and a mounted circular saw with three directional movement controls.
Abstract: A new type of apparatus for cutting 100–200µ sections of bone is described. This apparatus consists of an especially designed bone vise and a mounted circular saw with three directional movement controls. The saw is driven by one D.C. motor and its vertical movement assembly is driven by another. Their speeds can be regulated individually. The bone sections may be made serially at intervals of 0.3 to 0.5 mm. and bones as large as 2 cm. in diameter or longitudinal slabs of larger bones may be sectioned.
TL;DR: The dense connective tissues of the mouse and cat are selectively stained following the vital introduction of chlorazol fast pink, which parallel that of trypan blue, vitally as well as in vitro.
Abstract: The dense connective tissues of the mouse and cat are selectively stained following the vital introduction of chlorazol fast pink. In addition, the newly formed areas of bone and of cartilage are intensely stained, while other areas of these two tissues are unstained. The staining reactions of this dye parallel that of trypan blue, vitally as well as in vitro.
TL;DR: By this method it has been demonstrated that the Feulgen hydrolysis removes cytoplasmic and nucleolar RNA.
Abstract: The technics generally used for preparing root tip cells for microscopical examination destroy mitochondria and other cytoplasmic particles and remove lipoidal material. Fixation in a bichromate solution followed by treatment with osmic acid preserves these granules through normal embedding procedures (cf. Zirkle, 1929; Newcomer, 1940); also fixation in neutral formalin and embedding in the water-soluble wax, Aquax, may completely preserve lipoidal matter, and thus the mitochondria. The separation of cells in squash preparations usually entails acid hydrolysis of the intercellular cement. Treatment for one hour with a 5% solution of a commercial pectinase powder in a 1% aqueous solution of peptone allows good separation of cells of bean root tips fixed in acetic-alcohol. By this method it has been demonstrated that the Feulgen hydrolysis removes cytoplasmic and nucleolar RNA. To preserve the mitochondria it is advisable to immerse the bean roots in a 5% aqueous solution of polyvinyl alcohol for 24 hours a...
TL;DR: Iodoacetamide was the most effective sulfhydryl inhibitor as demonstrated by indicator reduction, and the reduction of the indicators by oxidized sulfHydryl compounds in the presence of potassium cyanide closely paralleled the Reduction of the sarrte indicators by reduced sulfhydyl compounds.
Abstract: There was considerable variation between the sulfhydryl induced in vitro reduction of the oxidation-reduction indicators (2,3,5-triphenyltetrazolium chloride, 2,3-diphenyl 5-methyl tetrazolium chloride, neotetrazolium chloride, neotetrazolium phosphate-2B, blue tetrazolium, tetrazolium violet, potassium tellurite, methylene blue, and resazurin). The neotetrazolium salts and potassium tellurite showed the greatest reducing activity. The reduction of the indicators by oxidized sulfhydryl compounds in the presence of potassium cyanide closely paralleled the reduction of the sarrte indicators by reduced sulfhydryl compounds. Iodoacetamide was the most effective sulfhydryl inhibitor as demonstrated by indicator reduction.
TL;DR: The method describes an adaptation of a metallurgical procedure whereby dry, calcined bone may be simultaneously infiltrated and embedded in a transparent plastic, Transoptic, and then ground to the desired thinness for microscopic observation with transmitted light.
Abstract: The method describes an adaptation of a metallurgical procedure whereby dry, calcined bone may be simultaneously infiltrated and embedded in a transparent plastic, Transoptic, and then ground to the desired thinness for microscopic observation with transmitted light. A 2 mm.-thick specimen of bone, ground smooth on one side, is placed ground side up in a 1″ mold assembly of a metallurgical specimen mount press. About 5 ml. of the plastic medium is added, the temperature raised to 130° C, and the pressure raised to 100 pounds. When 130° C. is reached, the heater is disconnected, the pressure immediately raised to 3500 pounds and maintained at that level until the mold cools to 68° C. The pressure is then released and the 5 mm.-thick plastic disc, with the embedded specimen therein, expressed from the mold. Grinding, as well as polishing, is dry; abrasives in fluid media are not used. The disc and specimen are coarse ground on #340 grit dry silicon carbide paper until histological details begin to appear. F...
TL;DR: An aqueous histologic and cytologic mounting medium containing polyvinyl alcohol, cadmium iodide and fructose is described, used in the preparation of permanent slides and may be applied to sections stained and rinsed in water or in any concentration of ethyl alcohol.
Abstract: An aqueous histologic and cytologic mounting medium containing polyvinyl alcohol, cadmium iodide and fructose is described. It is used in the preparation of permanent slides and may be applied to sections stained and rinsed in water or in any concentration of ethyl alcohol. The medium forms a hard, tough, quick-drying film which adheres well to the slide and cover glass. It is nonfluorescent, clear, optically homogeneous, and isotropic; it does not admit bubbles under the cover glass or crystallize on drying. It prevents or minimizes the bleeding of many stains from the sections; a list of 60 dyes, mostly basic, is given. The fresh medium has a pH of 4.4. The refractive index of the fresh medium is nD20 1.4674; the dried film is nD 1.6020 which may be lowered to nD 1.5150 by decreasing the cadmium iodide in the formula. The viscosity at 25°C. is 7,299 centipoises. The medium has the following composition: distilled water, 40 g.; cadmium iodide, 34 g.; polyvinyl alcohol (Elvanol 51–05, du Pont's low viscos...
TL;DR: Studies of the distribution, abundance, size and shape of sebaeous glands are most readily made with relatively permanent whole mounts of skin samples which are fixed in 10% formalin, washed, stained 8–16 hours with oil blue N, mounted on slides in glycerol gelatin.
Abstract: Studies of the distribution, abundance, size and shape of sebaeous glands are most readily made with relatively permanent whole mounts of skin samples which are: fixed in 10% formalin, washed, stained 8–16 hours with oil blue N (2 parts sat. oil blue N in 60% isopropanol: 1 part water), washed, dissected free of obscuring connective tissue, washed, and mounted on slides in glycerol gelatin. After 2 or 3 days, the edges of the cover glass are sealed with clear lacquer.
TL;DR: Pararosanilin hydrochloride or pararosAnilin base was purified by suspending 20 g.
Abstract: Pararosanilin hydrochloride or pararosanilin base was purified by suspending 20 g. of the dye in 400 ml. of water, acidifying with 50 ml. of 2N HCl and adding 4—5 g. of decolorizing charcoal. The mixture was then heated to boiling and boiled for 2 minutes. The entire mixture was transferred to a large, covered, fluted filter, and the filtrate allowed to stand overnight while the pararosanilin hydrochloride precipitated. The pararosanilin hydrochloride was filtered off, resuspended in 100 ml. of ether-alcohol (10:1) and shaken for 3 to 5 min. The ether-alcohol suspension was filtered and the pararosanilin hydrochloride washed repeatedly on the filter with ether until the ether wash was no longer colored. It was then washed several times with distilled water (total volume 400 ml.), dried in vacuuo over concentrated sulfuric acid, ground to a fine powder and stored in a dark brown bottle.
TL;DR: Two modifications of the method are described: Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear.
Abstract: Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.