Showing papers in "Biotechnic & Histochemistry in 1970"
TL;DR: The FCR provides an effective method for assessing pollen quality and is primarily one for the integrity of the plasmalemma of the vegetative cell, which is likely to be closely correlated with viability.
Abstract: Viable pollen grains immersed in a solution of fluorescein diacetate, made up in a concentration of about 10−6 M in sucrose of suitable tonicity (usually about 0.5 M), rapidly accumulate free fluorescein, which can be detected by its fluorescence. This fluorochromatic reaction (FCR) probably depends upon the entry of the nonpolar substrate into the vegetative cell where it is hydrolyzed by esterase to give the polar product, fluorescein, which is retained by the cell membrane. The test is primarily one for the integrity of the plasmalemma of the vegetative cell. Since this integrity is likely to be closely correlated with viability, the FCR provides an effective method for assessing pollen quality.
874 citations
TL;DR: The silver impregnation method of Fink and Heimer has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide and gave results comparable to those obtained on unmounted frozen sections of formal in-fixed material.
Abstract: The silver impregnation method of Fink and Heimer (Brain Res., 4: 369-74, 1967) has been used on cryostat sections of both fresh frozen and formalin-fixed brain tissue mounted on slide. The fixed brains were soaked in 25% sucrose for 2-3 days before freezing. The slides used for mounting were dipped in a 0.5% aqueous gelatin solution containing 50 mg of chrome-alum per 100 ml, drained and allowed to dry in a vertical position. Sections of fresh tissue were fixed for 16 hr in a 10% formalin solution buffered with phosphates to pH 7.0. Staining was carried out according to the Fink-Heimer procedure II and gave results comparable to those obtained on unmounted frozen sections of formalin-fixed material
90 citations
TL;DR: The water-soluble, nonfluorescent dye predominating in acid solutions became deprotonized, lipophilic and fluorescent under neutral and alkaline conditions, apparently dependent upon their composition.
Abstract: Aqueous 0.1% neutral red adjusted to pH 6.5, or mixed 1:1 with seawater, produced conspicuous fluorescence in plant and animal fats while acting as a conventional stain for acidic groups including volutin. The water-soluble, nonfluorescent dye predominating in acid solutions became deprotonized, lipophilic and fluorescent under neutral and alkaline conditions. Neutral dye crystals fluoresced dull red, whereas the fluorescence was either bright lemon yellow or blue-green in lipids, apparently dependent upon their composition.
83 citations
TL;DR: Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S; in all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rats, they displayed more affinity for eOSin and were reddish.
Abstract: Air-dried smears of saline suspensions of mammalian spermatozoa were stained for 10-60 min at room temperature in a mixture of eosin Y, fast green FCF, and naphthol yellow S (0.1% w/v, each dye) in 1.0% aqueous acetic acid. They were then blotted, rinsed in 1.0% acetic acid until no more dye was removed (0.5-1.5 min), blotted and allowed to dry completely, rinsed in xylene and mounted in synthetic resin. In all species examined except the rat, acrosomes were stained greenish blue to bluish green or blue depending on their thickness; in the rat, they displayed more affinity for eosin and were reddish. In all species, spermatozoan nuclei were strongly stained by naphthol yellow. In intact sperm heads, postnuclear cap had a yellowish green appearance. Midpieces of rodent spermatozoa, especially those of younger gametes, were stained bright red while those of ejaculated bull and rabbit spermatozoa were stained blue-green. Cytoplasmic droplets associated with rodent spermatozoa were consistently stained a dark...
71 citations
TL;DR: Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues and many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze- sectioning.
Abstract: Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or ...
56 citations
41 citations
TL;DR: Tissue which had been fixed in 4% glutaraldehyde and postfixed in 2% OsO4 was subsequently treated with p-phenylenediamine, either in the block prior to embedding in paraffin or Epon or, in the case of Epon-embedded material, after sectioning for light microscopy.
Abstract: Tissue which had been fixed in 4% glutaraldehyde and postfixed in 2% OsO4 was subsequently treated with p-phenylenediamine, either in the block prior to embedding in paraffin or Epon or, in the case of Epon-embedded material, after sectioning for light microscopy. The p-phenylenediamine was best used as a 0.8-1% solution in 70% alcohol. The p-phenylenediamine caused a very considerable intensification of staining of any cell components; this intensification of staining was particularly marked in the case of the lipid granules of renal medulla.
35 citations
TL;DR: Growing oocytes of the marine crab Cancer pagurus L. contain different kinds of yolk inclusions that presumably are lipids and the lipid character of these granules was demonstrated by extracting them from the epon-embedded ultrathin sections by using the method of Mayor et al.
Abstract: Growing oocytes of the marine crab Cancer pagurus L. contain different kinds of yolk inclusions. Some of them are strongly blackened by OsO4 and presumably are lipids. The lipid character of these granules was demonstrated by extracting them from the epon-embedded ultrathin sections by using the method of Mayor et al. (J. Biophys. Biochem. Cytol., 9:909, 1961) originally recommended for the removal of epoxy resin from sections intended for light microscopy. This technique was also applied to liver tissue of mouse with successful result.
33 citations
TL;DR: Fibrin clots were used for suspensions; fibrin films for preserving in situ characteristics of conidial chains, delicate fruiting structures, and cellular arrangements of mycetozoa and fungi.
Abstract: Fibrin clots were used for suspensions; fibrin films for preserving in situ characteristics of conidial chains, delicate fruiting structures, and cellular arrangements of mycetozoa and fungi. A solution containing fibrinogen, 300–350 mg; Na-citrate, 160 mg; and NaCl, 850 mg per 100 ml, was mixed immediately before use, and in the amount needed, with an equal volume of a thrombin solution containing 5–10 units/0.l ml of sterile glass-distilled water. In these proportions, a clot formed within 20–30 sec. Washed suspensions, pelleted by centrifugation, were incorporated into the forming clot by twisting a sharpened applicator stick, first in an overlying activated fibrinogen solution, and then into the pellet until the cell mass was entrapped. Films were prepared either by spreading the activated solution on a coverslip onto which a piece of natural substratum or of agar bearing the desired structures was impressed, or by dropping the activated solution directly on the structures' support.
31 citations
TL;DR: The behavior of the dye toward several phospholipids suggests that the phosphate groups are essential to the binding reaction and that the quaternary amine of phosphatidyl choline may interfere with the formation of isopropanol-insoluble complexes.
Abstract: A determination of the selectivity and approximate stoichiometry of Luxol Fast Blue ARN by known chemical compounds showed that phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl inositol bound the dye with an apparently stoichiometric ratio of 1 dye molecule to 2 molecules of lipid. Phosphatidyl choline, sphingomyelin, and palmitic acid showed a much weaker reaction. Of these, phosphatidyl choline bound the least amount of dye; about 1 dye molecule per 13-20 lipid molecules. Glycerides, methyl and cholesteryl esters of fatty acids, cholesterol, cerebrosides, and oleic acid gave negative results, as did a variety of low molecular weight substances, including ethanolamine, choline, inositol, and serine. Such negative results indicate that no isopropanol-insoluble complexes were formed with the dye. The behavior of the dye toward several phospholipids suggests that the phosphate groups are essential to the binding reaction and that the quaternary amine of phosphatidyl choline may interfere wit...
22 citations
Journal Article•
TL;DR: The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules and specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R to enhance visibility of unstained starch grains.
Abstract: The main constituents of wheat flour and many wheat flour products are wheat protein (gluten) and starch granules. The specific staining of the protein present was effected by 10 min in 0.1% aqueous ponceau 2R (C.I. No. 16150) acidified with 3—4 drops of 1 N H2SO4 per 50 ml of staining solution, followed by rinsing in 2 changes of distilled water, dehydrating, clearing and mounting in a resinous medium in the normal way. Staining of starch was as follows: sections or flour smears were brought to water, treated for 10 min in a protein-blocking reagent (Taninol ADR—Imperial Chemical Industries—used in 1% aqueous solution) rinsed, then stained for 3 mins in 0.5% aqueous chlorazol violet R (C.I. No. 32445) or for 10 min in either 0.5% aqueous chlorazol violet N (C.I. No. 22570), or chlorazol black E (C.I. No. 30235). Staining was followed by thorough rinsing, normal dehydration and clearing and mounting in a medium of R.I. about 1.49 to enhance visibility of unstained starch grains. The methods are applicable...
TL;DR: This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation and apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure.
Abstract: The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl2, the yeast cells were postfixed in 3% OsO4 at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure
TL;DR: Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.
Abstract: Aldehyde fuchsin, pseudoisocyanin and toluidine blue, histochemical dyes reported to be specific for insulin–containing granules of the pancreatic beta cell, were applied to insulin fixed in polyacrylamide gel by disc electrophoresis. Two major and four minor bands were resolved as demonstrated by staining with amidoschwarz; only the two major bands, were stained by aldehyde fuchsin. The addition of serum did not affect this reaction. Serum or insulin components gave no metachromatic reactions to the other stains. Under the conditions applied, aldehyde fuchsin is the only one of these dyes specific for insulin in this, system, but this stain is not sufficiently sensitive to detect normal serum levels of the hormone.
TL;DR: The use of Morin for fluorescent localization of aluminum in plant tissues was discussed in this paper, where it was shown that the Morin method can be used to detect aluminum in tissue.
Abstract: (1970). The Use of Morin for Fluorescent Localization of Aluminum in Plant Tissues. Stain Technology: Vol. 45, No. 6, pp. 301-303.
TL;DR: Animals injected with impure thymidine-3H, containing self-radiolysis decomposition products due to storage, led to significant alteration in the autoradiographic pattern, particularly those prepared by the freeze-dried method which show only 52% of the activity over the nucleus.
Abstract: Intestinal tissue from animals injected with tritium-labeled thymidine were examined by two autoradiographic methods. Conventional autoradiograms using fixed, solvent-dehydrated, paraffin-embedded tissues, and wet mounted, were compared with an autoradiographic method designed for localizing diffusible substances utilizing dry-mounted, freeze-dried frozen sections. The autoradiograms prepared by conventional methods show more than 95% of the activity located over the nucleus while, autoradiograms of intestinal tissue from the same animal prepared by the freeze-dried method shows only 88% of the activity over the nucleus. Animals injected with impure thymidine-3H, containing self-radiolysis decomposition products due to storage, led to significant alteration in the autoradiographic pattern, particularly those prepared by the freeze-dried method which show only 52% of the activity over the nucleus. Thus, conventional treatment of tissue extracted free unincorporated thymidine, metabolic products of thymidin...
TL;DR: A simple, rapid procedure was developed by which the quality of endosperm protein in cereal grains could be evaluated by microscopic observation of the subcellular protein structure by identifying sites of prolamine.
Abstract: A simple, rapid procedure was developed by which the quality of endosperm protein in cereal grains could be evaluated by microscopic observation of the subcellular protein structure. Kernels with vitreous endosperm were sectioned without pretreatment at 3-4 μ with a glass knife. Floury endosperm tissues were fixed in 10% glutaraldehyde, pH 7.4, for 16 hr at 5 C, and boiled to gelatinize the starch. After drying, this tissue was sectioned as for vitreous endosperm. Sections were mounted on gelatin-coated slides and destarched with a-amylase. To identify sites of prolamine, alcohol-soluble protein was extracted for 1 hr at about 70 C with 80% ethanol. The subcellular proteins were stained with either iodine vapor or various organic dyes. Proteins were differentiated by a combination of staining and mounting in selected high refractive index liquids.
Journal Article•
Journal Article•
TL;DR: This technique has been developed especially to stain sensory receptors which have been localised intramuscularly by electrophysiological means and can then be teased out and mounted in glycerol, under cover glasses ringed with a waterproof cement.
Abstract: This technique has been developed especially to stain sensory receptors which have been localised intramuscularly by electrophysiological means. Rat intertransverse caudal muscles, removed immediately after death, are fixed for 24 hr in a freshly prepared mixture of absolute ethyl alcohol, 4.5 ml; distilled water, 5 ml; and concentrated HNOa, 0.1 ml. After a further 24 hr in 10 ml of absolute ethyl alcohol containing 0.1 ml of ammonia solution (sp. gr. 0.88), the muscles are washed in distilled water for 30 min and placed in full strength pyridine for 2 days. They are then washed for 24 hr in distilled water (changed 5-8 times) and left in 2% AgNO3, in the dark for 3 days at 25 C. Following reduction in 10 ml of 5% formic acid containing 0.4 gm of pyrogallol for 6-24 hr, the specimens are washed briefly in distilled water and stored in pure glycerol. The nerve endings can then be teased out and mounted in glycerol, under cover glasses ringed with a waterproof cement. The advantage of this method is that i...
TL;DR: Heinz bodies stained deep purple and contrasted well with the yellow-orange to blue-green cytoplasm and the reticular material in reticulocytes did not stain in 2 min but could be stained by allowing the stain to act 5 min.
Abstract: Equal volumes of heparinized or EDTA-treated blood and a 0.5% solution of rhodanile blue (E. Gurr, Michrome No. 1156) in 1% NaCl were mixed and allowed to stand for 2 rain. Thin smears were then prepared, air dried and examined under oil. Heinz bodies stained deep purple and contrasted well with the yellow-orange to blue-green cytoplasm. Durable mounts could be made by applying a cover glass with a resinous mediiun to the dry smear (D. P. X. was used). The reticular material in reticulocytes did not stain in 2 min but could be stained by allowing the stain to act 5 min.
TL;DR: The emission spectra of the fluorochrome, SITS, are stable in the pH range of 4.5—12.0 and may be useful as a vital dye since it distinguishes between living and dead cells.
Abstract: The emission spectra of the fluorochrome, SITS, are stable in the pH range of 4.5—12.0. The wavelength range giving maximum excitation was 340-360 mμ; range of maximum emission, 415-420 mμ In the animal cell types tested, HeLa, Chang liver, mouse L, and monkey kidney, all cellular membranes stained, but cell walk of plant tissue (Allium cepa) did not. The staining solution was 5 mg/ml of SITS in Earle's balanced salt solution. The preparations were mounted and viewed microscopically in this solution or, after staining and protracted storage in 9:1 methanol-formalin mixture they were mounted and viewed in this fixative. SITS may be useful as a vital dye since it distinguishes between living and dead cells.
TL;DR: Pyrenoids but not nucleoli are clearly stained with propionocarmine after a special fixation, and the pyrenoid is distinguished as the largest dark purple, intracellular body that stains homogeneously.
Abstract: Pyrenoids but not nucleoli are clearly stained with propionocarmine after a special fixation. The pyrenoid is distinguished as the largest dark purple, intracellular body that stains homogeneously. The general procedure is as follows: unicells and filaments are collected by centrifugation and any liquid discarded; cells are fixed 5 rain in a mixture of 10 volumes of 50% ethanol, 10 of concentrated HC1, and 5 of Clorox; washed in 95% ethanol 3 times or more to remove all chlorophyll and fixative; mordanted for 5 min with a suitably adjusted concentration of ferric propionate in 50% propionic acid (reddish orange in color); stained 5 min in 0.5% carmine in 50% propionic acid heated in a boiling water bath. The material is mounted in 50% propionic acid. The results are reproducible when material is processed in centrifuge tubes and when the optimum time in the mordant and stain is determined for a particular species and then repeated accurately. This technique was successful in staining pyrenoids of all of t...
TL;DR: The degassed Schiff reagent contained only traces of-SO3H as judged from its minimal absorbance between 280 and 295 nm, and the UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical.
Abstract: Ultraviolet (UV) absorption (200-330 nm) for 0.5 mg/ml aqueous solutions of basic fuchsia unadjusted and those adjusted to pH 0, 1.5, 8.5, and 11 were determined as well as spectra in the visible range (400-675 nm) for solutions with pH 1.9, 2.8, 3.9, 4.7, 5.5, 5.9, 6.5, 7.5, 9.3, 10.4, and 11. The UV absorbance of degassed Schiff reagent containing 1 or 0.5 mg dye/ml, and that of this reagent adjusted to pH 1.5, 2.3, 3.1, 4.5, 6.0, 7.1, and 8.4 were obtained for comparison. The progressive reaction of formalin with degassed Schiff reagent, followed spectrometrically for 2.5 hr, required 2 hr to reach completion. The degassed Schiff reagent contained only traces of-SO3H as judged from its minimal absorbance between 280 and 295 nm. The UV absorption of this reagent and basic fuchsin in 1 N HCl were found to be identical. The absorbance is that of basic fuchsin reduced by the addition of Cl− or SO3H− to the central methane carbon and H to the amino groups, therefore the leuco structure of basic fuchsin so r...
Journal Article•
TL;DR: One-day-old mycelia of Volvariella volvacea, grown on water-permeable cellophane membrane applied to an agar medium, were stripped from the medium and fixed 1 hr in 3% glutaraldehyde followed by 2 hr in 1% OsO4.
Abstract: One-day-old mycelia of Volvariella volvacea, grown on water-permeable cellophane membrane applied to an agar medium, were stripped from the medium and fixed 1 hr in 3% glutaraldehyde followed by 2 hr in 1% OsO4. The specimens were washed in water, dehydrated with acetone and embedded in Vestopal W. The flat end of the precast tissue carrier was placed on the top (tissue side) of the specimen in a polyethylene dish. The preparation was first allowed to harden 12 hr at 50 C. After hardening was completed at 60 C., the block was removed, the cellophane stripped off, and the face of the block trimmed for sectioning. Since the mycelium had grown as a thin flat layer, the face of the block required little or no trimming, and sections in the plane of the mycelial filaments were readily obtained.
TL;DR: The Falck and Hi Harp technique for the cellular localization of catecholamines by formaldehyde-induced fluorescence was applied to rat, mouse, and Syrian hamster adrenal, and some medullary cells revealed an unexpected orange-brown fluorescence.
Abstract: The Falck and Hi Harp technique for the cellular localization of catecholamines by formaldehyde-induced fluorescence was applied to rat, mouse, and Syrian hamster adrenal. Some medullary cells revealed an unexpected orange-brown fluorescence. In guinea pig or rabbit adrenals, which store predominantly epinephrine, orange-brown fluorescence was not readily observed. It was found in Syrian hamsters only at the medullary periphery, where norepinephrine-producing cells are known to occur. Orange-brown fluorescence was depleted by administration of reserpine and intensified by nialamide plus DOPA. The same cell clusters which stained specifically for norepinephrine with ferric ferricyanide were found in adjacent sections to exhibit orange-brown fluorescence. Only by reducing the temperature of the formaldehyde reaction could sections of hamster adrenal showing only yellow-green fluorescence be obtained. These data suggest that the orange-brown fluorescence might result from' polymerization, oxidation, or both,...
TL;DR: A higher peroxidase activity was observed in the liver than in the muscle, and among the red and white fibres of muscle, the former were comparatively richer in the enzyme, which appeared to be mitochondrial.
Abstract: For the histochemical localization of a special ascorbic acid-free radical (AA.FR) peroxidase, frozen sections (10–15 μ) of fresh liver and striped muscle from pigeon and domestic fowl were immersed for 18–24 hr in an incubation medium consisting of: saturated aqueous o-dinitrobenzene, 5 ml; ascorbic acid 5 mg/ml, 5 ml: 3% H2O2, 1 ml; and citrate buffer pH 6.4–6.6, 2 ml. Sections were then rinsed with water, treated with 1% NH4OH for 2 min, rinsed again with water and mounted in glycerol jelly. The cells containing the AA-FR peroxidase acquired a deep purple color. Three controls consisted of: (i) replacement of AA with 5 ml of water; (ii) addition to the incubating medium of 5 ml of 0.002 M Na2S2O5; or (iii) addition of 2 × 10−2M catalase (from buffalo Liver—Biochemical Unit, V. P. Chest Instt. Delhi-7). A higher peroxidase activity was observed in the liver than in the muscle. Among the red and white fibres of muscle, the former were comparatively richer in the enzyme, which appeared to be mitochondrial...
TL;DR: The neutralized Schiff's reagent was satisfactory as an aldehyde reagent in the nucleal reaction on gut and, although giving a less intense reaction than the routine reagent on the gut and plasmal reaction on the aorta, was satisfactory here in respect to localization and thus to aldehydes specificity.
Abstract: A neutralized Schiff's reagent (pH 6.7) was prepared and used to investigate the role of the acidic nature of the routine Schiff's reagent (pH 2.6) in the plasmal reaction. The neutralized reagent was satisfactory as an aldehyde reagent in the nucleal reaction on gut and, although giving a less intense reaction than the routine reagent in the PAS reaction on the gut and plasmal reaction on the aorta, was satisfactory here in respect to localization and thus to aldehyde specificity. Control sections for the plasmal reaction of unfixed nerve and aorta gave positive results when placed in the routine Schiff's, this increasing with time left in the reagent. Similar control sections in the neutralized Schiff's reagent remained consistently negative even though left in this reagent for 0.5 hr. The positive reaction of such control sections is apparently due to acid hydrolysis of labile plasmalogens by the routine Schiff's reagent in myelin and elastin and not to the presence of “free” aldehydes in these tissue ...
TL;DR: Tissues from representative mammals, amphibia and invertebrates were fixed for 5-24 hr in either an aqueous solution of 8% p-toluene sulfonic acid (PTSA) or in 10% formalin to which 5 gm PTSA/100 ml had been added, and processed through embedding in polyethylene glycol 400 distearate in the usual manner.
Abstract: Tissues from representative mammals, amphibia and invertebrates were fixed for 5-24 hr in either an aqueous solution of 8% p-toluene sulfonic acid (PTSA) or in 10% formalin to which 5 gm PTSA/100 ml had been added, and processed through embedding in polyethylene glycol 400 distearate in the usual manner. Sections cut at 4-6 μ were floated on 0.2% gelatin containing 1.25% formalin, and spread and dried on slides at a temperature not exceeding 25 C. Wax was removed with xylene, and the sections brought to water through ethanol as usual. The working staining solution was made from three stock solutions: A. Chlorantine fast blue 2RLL, 0.5%; B. Cibacron turquoise blue G-E, 0.5%; C. Procion red M-P, 0.5%—each of which was dissolved in 98.5 ml of distilled water to which 0.5 ml of glacial acetic acid and 0.5 ml of propylene glycol monophenyl ether (a fungicide) had been added. For use, the three solutions were mixed in the proportions: A, 3; B, 4; and C, 3 volumes. Staining time was uncritical, 10-30 min usually...