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.     

Tissue 1,2-Glycol Localization with Phenyl Iodosoacetate

A solution of phenyl iodosoacetate in glacial acetic acid is capable of splitting the 1,2-glycol linkage in tissue sections to produce a histological picture with leucofuchsin resembling that obtained under similar conditions with lead tetraacetate.     

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.     

Modification of maize starch by thermal processing in glacial acetic acid

Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) methods were used to determine if corn starch–glacial acetic acid mixtures can be melted and thermally processed at reasonable temperatures. DSC studies showed that the melting temperature of dry starch was reduced from about 280 to 180°C in the presence of >30% acetic acid. Glass transition temperatures varied from 110 to 40°C at 15 and 45% acetic acid, respectively. XRD showed the loss of native starch crystallinity and the formation of V-type complexes. Addition of 10% water decreased the melting temperatures to 140–150°C while addition of a base (sodium acetate) had little effect. Some possible applications of processing starch in glacial acetic acid will be discussed.     

Preparing Serial Sections of Mature Corn and Wheat Kernels

Methods are described for preparing serial sections of paraffin-embedded mature corn and wheat kernels. Prior to embedding corn kernels are killed and fixed in formalin-aceto-alcohol (FAA), then steeped 5 days in 50% glycerol. After embedding by a special procedure, a thin slice is cut from one side of the kernel and the first few cell layers removed. The exposed surface is submerged in 20% glacial acetic acid in 60% ethanol for 2 or 8 days depending on the surface exposed, 2 days in air at 100% relative humidity at room temperature, and 2 days in air at 100% relative humidity at 8°C, successively. Wheat kernels, fixed in formalin-aceto-alcohol and embedded by the regular paraffin procedure, are similarly trimmed to expose a surface which is submerged in 20% glacial acetic acid in 60% ethanol for 2 days, 2 days in air at 100% relative humidity at room temperature and 2 days in air at 100% relative humidity at 8°C, successively. The corn and wheat kernels prepared by these methods give good serial sections when cut as thin as 14μ. The application of these methods to other seeds and caryopses is suggested.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

Staining Sex Chromatin; Biebrich Scarlet and Fast Green Fcf as a Mixture Versus Their Use in Sequence

Human skin was fixed in Davidson's solution (95% alcohol, 35; formalin, 20; glacial acetic acid, 10; and distilled water, 35—parts by volume) and sections prepared through paraffin embedding in the usual manner. Stock stains were: I(BS)—Biebrich scarlet, 1 gm in 100 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid and 5 ml of glacial acetic acid were added—and II(FG)—fast green, 0.5 gm in 85 ml of 50% alcohol to which 0.3 gm of phosphotungstic acid, 0.3 gm of phosphomolybdic acid, and 15 ml of glacial acetic acid were added. Experimental staining solutions were prepared in the following proportions of stock BS to stock FG—1:1, 2:1, 3:1, 1:2 and 1:3. Sections were brought to 50% alcohol and stained for 15, 20, 25 and 30 min in each of the five BS-FG mixtures, rinsed in 50% alcohol, then dehydrated in 70%, 95%, and absolute alcohol, 2 min each; cleared in xylene, and covered in balsam. The 2:1 (optimum proportion) combination of BS with FG, acting for 20 min, yielded 97% sex chromatin-positive nuclei in female material. If sections were stained in stock solution BS for 2 min, they could be differentiated by a 20 min treatment in the mordanting component of stock FG (without dye) to give a one-color stain. Such stains gave about the same percentage of sex chromatin-positive nuclei as those obtained by the regular two-color procedure. These modifications are simpler, more rapid, and yield results comparable to previously employed techniques.     

A Bodian method for mounted frozen sections.

For application of the Bodian method to frozen sections, cut frozen peripheral nerve or muscle at 10 mum and mount. Fix for 4 days in 18 parts 80% ethanol, 1 part 10% formalin, and 1 part glacial acetic acid. Fix central nervous tissue in the same mixture prior to freezing and sectioning, and after mounting postfix for 4 days. Impregnate by the Bodian procedure. The results equal Bodian stains of paraffin sections. The technique is simple and reliable. The use of 10 mum frozen sections produces little artifact and allows alternate serial sections to be stained with other techniques.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

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).     

Histochemical Use Of Lead Tetra-Acetate I. Cleavage Of 1-2 Glycols

Lead tetra-acetate acts specifically to split the carbon-carbon single bond of the 1,2-glycol linkage to produce aldehyde radicals which may then be demonstrated by means of leucofuchsin, 2,4-dinitrophenlyhydrazine, or p-nitrophenylhydrazine. Routinely prepared slide sections from tissues fixed in 10% formalin are run down to 95% alcohol, rinsed in glacial acetic acid and then treated for 2 minutes in a saturated solution of lead tetra-acetate in glacial acetic acid with 5 g. of potassium acetate added for each 100 ml. of reagent. The sections are then washed in distilled water and placed in leucofuchsin for 10 minutes, or in a saturated 30% alcoholic solution of p-nitrophenylhydrazine for 5 minutes or 2,4-dini-trophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 minutes if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofuchsin or yellow by the nitrophenylhydrazines.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

A Controllable Carmine Technic for Plants with Small Chromosomes

Anthers of small chromosome plants (Antirrhinum, Brassica, Capsicum etc.) were fixed 12 hours or longer at 0-3° C. in: ferric acetate in glacial acetic acid (sat. soln.), 1 part; absolute alcohol, 3 parts. They were transferred to: ferric acetate (sat. soln.) in 45% acetic acid, 3 parts; 45% acetic acid, 5 parts; 1% formalin (aq.), 2 parts, and allowed to remain 5-15 minutes at room temperature for mordanting. The amount of iron introduced into the specimens was controllable by the time in the mordanting fluid. After rinsing the specimen in 45% acetic acid and macerating in a drop of Belling's acetocarmine on a slide, a cover slip was applied followed by warming and pressing with blotting paper to flatten the pollen mother cells and expel excess stain. Preparations stored temporarily by sealing the edges of the cover slip with rubber solution were best made permanent by removing the cover slip after 1-2 days, dehydrating and mounting in euparal.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Golgi Method without Osmium Tetroxide for the Study of the Central Nervous System

A variant Golgi technique was developed that consisted of substituting osmium tetroxide with formaldehyde as the initial fixative in intracardiac perfusion, along with the addition of glacial acetic acid to the chromating fluid. This procedure avoids disposal of dangerous waste substances into the environment. Other advantages include 1) reduction of cost, danger to lab workers, and risk of disruption of the tissue slices during their handling by eliminating the osmium tetroxide, 2) clear tissue background, 3) greater quantity of impregnated neurons than in the classical procedure, with distinct morphological details easily identified even in gross sections and 4) reduction in processing time.     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Histochemical Analysis of Microdissected Nephrons: Enzyme Stains

Enzyme stains were done on 120-160 μ, sections of fresh-frozen human kidney obtained at autopsy or biopsy. These sections were then either fixed in a 3:7 mixture of ethanol and glacial acetic acid and macerated in collagenase, or fixed in 10% formalin and macerated in concentrated HCl. Following maceration by either method, the sections were washed in water and microdissected. With collagenase maceration, good results were obtained for dehydrogenase methods using tetrazolium salts. HCl maceration caused loss of formazans, but did not destroy the colored products of staining procedures for alkaline and acid phosphatase, aliesterase, and cytochrome oxidase.