A New Method Using a Modified Mayer's Hemalum at pH 6 for Demonstrating Mucous Neck Cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

The Use of Bismarck Brown Y in some new Stain Schedules

The staining quality of Bismarck brown Y may be improved and sterility maintained by adding 5% phenol to a 1% aqueous solution. Use the phenolic Bismarck brown in combination with iron alum hematoxylin except for stripped epidermis in the following procedures:

Stem and Root Schedule: Mordant sections from water in 4% iron alum for 10 minutes. Rinse in distilled water and stain in 0.5% aqueous hematoxylin for 1 minute or until darkly stained. Rinse in distilled water and destain in 2% iron alum until a gray color appears. Rinse thoroly in distilled water and intensify hematoxylin by transferring sections to 0.5% aqueous lithium carbonate until the desired black color appears. Rinse thoroly in distilled water and stain for 1-5 minutes in phenolic Bismarck brown. Rinse in distilled water, dehydrate successively in 30, 50, 70, 95 and 100% alcohol. Clear in methyl salicylate for 5 minutes, then to xylene for 3-5 minutes, and mount in balsam.

Middle Lamellae in Wood: Destain more thoroly in 2% iron alum than for the general stem and root schedule, and intensify in lithium carbonate for a longer period (about 1 hour).

White Potato Tuber Sections: Modify above schedule by reducing time of destaining in 2% iron alum to about 30-60 seconds and intensify hematoxylin until starch grains appear bluish in color. Stain in phenolic Bismarck brown for 1-2 minutes.

Wheat Grain Sections: Fix grain for sectioning when in “dough” stage. Use schedule the same as for potato tuber except for reducing time of staining in phenolic Bismarck brown to about 45 seconds.

Tradescantia zebrina Epidermis: Strip epidermis from leaf while submerged in water. Fix in 100% alcohol 10 minutes, pass thru 95, 70, 50, 30, and 10% alcohol to water. Stain in phenolic Bismarck brown for 10-20 minutes. Dehydrate, clear in methyl salicylate and mount in balsam.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Picric Acid as A Destaining Agent for Iron Alum Hematoxylin

A saturated aqueous solution of picric acid is used to differentiate paraffin sections and smeared pollen-mother-cells stained in Heidenhain's and Delafield's hematoxylins. The method proceeds as usual, except that the iron alum in the destaining process is replaced by picric acid. Mixtures of picro-sulfuric acid and dilute Delafield's hematoxylin and mixtures of picric acid and aqueous hematoxylin have also been tested, of which the latter yields the better result, but is not as good as the other method described here.     

Destaining Agents for Iron Alum Hematoxylin

Detailed directions for using various destaining agents after iron alum hematoxylin are given. A saturated aqueous solution of picric acid is recommended for bringing out nuclei in whole mounts of protozoa. Differentiation with a mixture of 1 part 30% hydrogen peroxide, and 2 parts, 95% alcohol, is useful for cytoplasmic structures. Greater reliability and precision are claimed for these methods as compared with the conventional differentiation in iron alum.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Mallory's Connective Tissue Stain Following Hematoxylin

The following combination of hematoxylin with Mallory's connective tissue stain is useful in bringing out nuclei as well as in differentiating tissue:

Slightly overstain in Mayer's hematoxylin (50 g. potassium alum and 0.2 g. sodium iodate added to 1 liter 0.1% aqueous hematoxylin). Wash; and stain 30 seconds to 1 minute in 0.04% aqueous acid fuchsin-Stain 4 minutes in: 0.5 g. anilin blue and 2 g. orange G dissolved hi 100 cc. of 1% aqueous phosphomolybdic acid. Pass thru 95% alcohol to absolute; clear in xylol and mount in balsam.     

In Toto Staining and Preservation of Peripheral Nervous Tissue

A simple quantitative modification of the in toto staining technique for nervous networks of Sihler is described. The results are demonstrated on the innervation pattern of the hard palate of the rat. Formalin fixed hard palates of rat were macerated and bleached in an aqueous solution of 3% potassium hydroxide with a few drops of 3% hydrogen peroxide added. Thereafter, the specimens were decalcified in Sinter's solution I (1 part glacial acetic acid, 1 part pure glycerin and 6 parts 1% chloral hydrate), the process being controlled by radiography. The specimens were next stained in Sutler's solution II (1 part Ehrlich's hematoxylin, 1 part pure glycerin and 6 parts 1% chloral hydrate). After staining, the non-nervous tissues were destained in Sihler'g solution I. Destaining was checked microscopically and was stopped before the finest branches of the nerves began to fade. The specimens were then washed in a weak aqueous solution of lithium carbonate and cleared in increasing concentrations of glycerin. Good visualization of nervous structures and a deep field of observation resulted; orientation of the peripheral nerves with respect to surrounding structures was readily seen and a three-dimensional image of the nervous networks was obtained.     

In toto staining and preservation of peripheral nervous tissue

A simple quantitative modification of the in toto staining technique for nervous networks of Sihler is described. The results are demonstrated on the innervation pattern of the hard palate of the rat. Formalin fixed hard palates of rat were macerated and bleached in an aqueous solution of 3% potassium hydroxide with a few drops of 3% hydrogen peroxide added. Thereafter, the specimens were decalcified in Sihler's solution I (1 part glacial acetic acid, 1 part pure glycerin and 6 parts 1% chloral hydrate), the process being controlled by radiography. The specimens were next stained in Sihler's solution II (1 part Ehrlich's hematoxylin, 1 part pure glycerin and 6 parts 1% chloral hydrate). After staining, the non-nervous tissues were destained in Sihler's solution I. Destaining was checked microscopically and was stopped before the finest branches of the nerves began to fade. The specimens were then washed in a weak aqueous solution of lithium carbonate and cleared in increasing concentrations of glycerin. Good visualization of nervous structures and a deep field of observation resulted; orientation of the peripheral nerves with respect to surrounding structures was readily seen and a three-dimensional image of the nervous networks was obtained.     

Method of combined detection of nerve fibers and blood vessels in nerve trunks]

A new method for investigation of neuro-vasal relationships in nerve conductors is proposed based on a combination of the fine injecting of their blood vessels and staining the nerve fibres. The vessels are injected with the chloroform emulsion of finely ground Paris blue (5-10 g per 100 g of solvent). Pieces of nerves 0,5 cm thick are fixed in 12% neutral formalin for 3 days, kept in a dark vessel in Weigert's mortant for 5 days, dehydrated and imbedded in paraffin. Thin slices 4-5 micronkm thick are stained in hematoxylin after N. N. Kulchitski, differentiated for 2-12 h in the mixture containing 1% solution of potassium ferricyanide and saturated solution of lithium carbonate (1:10) and after passing through alcohols of increasing concentration and xylene imbedded in balsam. In the preparations the fibres of different caliber are stained grey and the vessels are stained blue.     

Modifications in Bowie's Staining of pepsinogen granules

Modifications of Bowie's technic for staining the pepsinogen granules of the body chief cells in the gastric glands, and a method of preparing the stomach of small rodents (e.g. the rat) for histo-topographical studies are described. The modifications involve counter-staining with aqueous alum hematoxylin, the use of the neutral synthetic mounting medium Permount, and changes in the timing of fixation, staining and differentiation.     

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.     

Loss of Silver Grains from Radioautographs Stained by Gallocyanin-Chrome Alum

Radioactive tissue sections covered with the film from Kodak Fine-Grain Autoradiographic Stripping Plate AR. 10 were stained with Ehrlich's hematoxylin or gallocyanin-chrome alum after exposure and photographic processing. Staining with gallocyanin-chrome alum at pH 1.7 and 2.4 dissolved the silver grains completely or almost completely in 1 to. Grains were quite visible after a 3 hr staining at pH 3.4, but a statistical analysis revealed a loss of grains, compared with unstained controls. Grains were also lost in slides immersed in solutions of gallocyanin alone at pH 2.5 for 24 hr but not in solutions of chrome alum alone, nor in some other alums. In sections stained 1 hr with Ehrlich's hematoxylin, the grains were not dissolved.     

Modifications in Bowie's Staining of pepsinogen granules

Modifications of Bowie's technic for staining the pepsinogen granules of the body chief cells in the gastric glands, and a method of preparing the stomach of small rodents (e.g. the rat) for histo-topographical studies are described. The modifications involve counter-staining with aqueous alum hematoxylin, the use of the neutral synthetic mounting medium Permount, and changes in the timing of fixation, staining and differentiation.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

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.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

An Evaluation and Modification of Cole's Hematoxylin

Consistency in staining with an alum hematoxylin is possible by the routine use of fresh staining solutions. A modification of Cole's hematoxylin is so easily prepared that fresh staining solutions present no problem. The staining solution consists of 100 ml 1.2% aqueous KA1(SO₄)₂ .12 H₂O, 1 ml 10% alcoholic hematoxylin and 2 ml 1% iodine. Mix, place in paraffin oven overnight and stain sections 5 minutes. The three solutions can be kept as stock solutions for years.     

Notes on Technic

Generally accepted staining methods using hematoxylin for identification of elastic tissues include Verhoeffs iron-hematoxylin (Verhoeff 1908), Herx-heimer's lithium carbonate-hematoxylin (Herxheimer 1886), Harris' aluminum chloride-hematoxylin (Harris 1902), and Petry's phosphomolybdic acid-hematoxylin (Pearse 1975). Since alum hematoxylin shows little or no affinity for elastic fibers, it is not used for staining elastic fibers.     

Localization of phospholipid-rich zones in rat gastric mucosa: possible origin of a protective hydrophobic luminal lining

Mammalian gastric mucosa is unusually hydrophobic or nonwettable, which may be an essential biophysical characteristic of the gastric mucosal barrier. Since this property may be attributable to an adsorbed layer of surface-active phospholipids (SAPL), we investigated the distribution of SAPL in rat oxyntic mucosa. Ferric hematoxylin (FH) and iodoplatinate (IP), selective histochemical stains for phospholipids (as confirmed by spot tests), were used to detect SAPL in frozen sections and aldehyde-fixed tissue, respectively. Using FH staining in conjunction with extraction procedures that either solvate or preserve SAPL, we determined that positive reactivity was the greatest in the apical third of the oxyntic mucosa between the glandular neck region and the surface. IP reactivity appeared to parallel the FH staining pattern. Mucous cells, especially the surface epithelial cells, were heavily stained. Electron microscopic examination revealed that these cells contain inclusion bodies associated with various subcellular organelles, e.g., nuclear envelope, endoplasmic reticulum, Golgi apparatus and its vesicles, and mucous secretory granules. Vesicles and myelin figures, which resembled those found in lung surfactant, were observed extracellularly in close association with the surface mucous cells. Our findings suggest that mucous cells are actively involved in synthesis and storage of SAPL, which may be an essential component of the stomach's protective hydrophobic lining.