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.     

Comparison of Various Oxidants for Alum Hematoxylin

Oxidation of 100 mg of hematoxylm in 40 ml of a 3% solution of EAI(SO₄),₂. 12H₂O with 20 mg iodine, 4.5 mg of NaIO₃. 3.5 mg of H₅IO₆, 3.5 mg of NaIO₄ or 9 mg KMnO₄ gives equivalent results as shown by both staining and absorbance measurements Placing the prepared solutions at 55 C for 18 hours increased absorbance by 1/4 to 1/3 over those kept at room temperature. Two months after preparation, solutions initially placed at 55 C for 18 hours, with no air space in the bottle, had not increased in absorbancy. In simiiar preparations kept at room temperature the absorbance increased from initial values to that of those that had been heated. Solutions in bottles with air space also showed this increase and also had a scum on the surface and sufficient precipitate to require filtering before use.     

A Hematoxylin Staining Mountant for Microorganisms

A gum arabic mounting medium containing iron-hemotoxylin is described. It is suggested for use as a one step process for making permanent slides of fungi and algae where a simple procedure is of importance in routine use, and where structures are being studied that may be damaged, or displaced, by transfer from one solution to another.     

Difficulties in Ferrous Iron Mordant Hematoxylin Staining and Some Hematoxylin Substitutes

A gradual deterioration of intensity of sequence ferrous sulfate hematoxylin staining was traced, after elimination of hematoxylin quality as a cause, to a deterioration of the metal salt, associated with caking of the crystals. Fresh samples were also partly caked and ineffective. Ferrous ammonium sulfate was found also subject to the same deterioration. Ferrous chloride freshly prepared as a 1 M solution from iron wire under anaerobic conditions at biweekly intervals proved to be satisfactory as a mordant source. Of several other mordant dyes tested: gallein, brazilin and chromoxane pure blue B were the best, but none was equal to good hematoxylin.     

苏木精染色液的改进和使用

报道了一种新的特制苏木精染色液的配制、使用方法、染色效果及其使用价值。实验表明,该染色液全面而明显地优于通用的Harris染色液。     

Hematoxylin Staining of Tissues Embedded in Epoxy Resins

Sections of 0.5-2 μ thickness are affixed to slides with albumen adhesive, thoroughly dried, and placed in xylene or toluene for 1 hr, then brought through ethanol to water. Sections of tissue fixed in O_sO₄ are treated first in 0.1% KMnO₄, then with 1.0% oxalic acid, and after rinsing, incubated at 60 C for 12-24 hr in hematoxylin (Harris's or Ehrlich's) and counterstained 10-15 min with 0.5% phloxine B. Permanent preparations are made by clearing and mounting in a synthetic resin. The method requires only easily available reagents and is suitable for routine processing of epoxy sections.     

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.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

A Stable, High-Contrast Mordant for Hematoxylin Staining

Small, quantities of sulfuric acid will stabilize iron mordants, used in hematoxylin staining, by preserving these solutions against oxidation. The presence of acetic acid in the mordant improves the specificity of the stain. A stable, high-contrast mordant is obtained when both acids are combined with ferric-ammonium sulfate. This mordant, used in combination with fresh alkaline solutions of hematoxylin, has been found especially effective in the staining of certain nuclear and cytoplasmic components of plant cells.     

Delafield'S Hematoxylin and Safranin for Staining Plant Materials

An improved schedule is suggested for staining plant materials in Delafield's hematoxylin and safranin. Tissues are stained first in Delafield's hematoxylin. A short bath in acidulated water (1 or 2 drops concentrated HCl to 100 cc.) removes objectionable precipitates, and at the same time serves as a destaining agent. The acid bath must be followed quickly by a thoro wash in tap water, or dilute lithium carbonate solution, to restore the original dark blue color (made reddish in the acid bath) of the hematoxylin and to “set” the stain. Once the hematoxylin solution is satisfactory, none of the reagents ordinarily used will remove it—unless they contain acid. Tissues are counterstained in rapid safranin (5 drops analin in 100 cc. of 1% safranin 0 in 50% ethyl alcohol); this materially lessens the time necessary for staining. The safranin is de-stained in 50% ethyl alcohol (which does not affect the hematoxylin) until sharp differentiation is secured. If destaining is too slow, or differentiation poor, a quick rinse in acidulated 50% alcohol usually sharpens contrast of the stains. This must be followed quickly by a wash in 50% alcohol containing lithium carbonate to neutralize the acid. Dehydrate, and mount as usual. This schedule allows each stain to be individually, and independently, controlled at the will of the operator.     

Block Staining of Mammalian Tissues with Hematoxylin and Eosin

Various mammalian tissues were stained en bloc with hematoxylin and eosin after fixation and prior to embedding in paraffin wax and sectioning. The choice of fixative is important and best results are obtained using Worcester's Fluid, a combination of saturated aqueous mercuric chloride, formaldehyde, and glacial acetic acid. After fixation, blocks of tissue up to 1.5 cm thick are stained for seven days in hematoxylin. Excess stain is removed by washing tissues in running water overnight. Tissue blocks then are dehydrated with graded concentrations of ethyl alcohols to 80% and counterstained, with further dehydration, in 0.5% spirit soluble eosin in 90% ethyl alcohol for five days. The tissue is subsequently transferred to 90% ethyl alcohol overnight to differentiate eosin staining; dehydration is completed in absolute ethyl alcohol. The blocks are cleared in cedarwood oil and briefly in xylene prior to embedding, sectioning, and mounting. Following removal of wax by xylene, coverslips are applied.

General morphological and histological features were particularly well differentiated and very selectively and reliably stained by this method.     

A Hematoxylin Staining Procedure for Maize Pollen Grain Chromosomes

Studies of postmeiotic chromosome behavior have been impeded by the thick exine and abundant starch grains of maize pollen. Staining pollen grain chromosomes with acetocarmine is tedious and gives inconsistent, often unsatisfactory results. A hematoxylin stain, used in conjunction with the clearing agent chloral hydrate, has been successfully used by the authors to stain chromosomes, nuclei and sperm cells of the maize pollen grain. An ethanol-formaldehyde fixing fluid is used to fix and preserve the pollen samples. The procedure, which is rapid and simple, gives excellent preparations with both fresh and fixed material. Stained preparations do not get darker with time, as is typical of other hematoxylin stained materials.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine, Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

The Staining of Brunner's Gland and Other Neutral Mucins by Carmine,Hematoxylin and Orcein in Alkaline Solutions

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Critical Staining of Pancreatic Alpha Granules with Phosphotungstic Acid Hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO₄ acidified with 0.3% (w/v) H₂SO₄, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.     

Staining Myelin, Elastic Fibers and Nuclei with Iron Hematoxylin

Two iron hematoxylin staining procedures were developed. Both use stable stock solutions and can be prepared volumetrically. The nuclear stain is progressive but differentiation is required for myelin sheath and elastic tissue staining. Histochemical procedures demonstrated that acid, hydroxyl, and aldehyde groups play no role in the staining but amine groups are essential. With both types of stains neither electrostatic bonding nor hydrogen bonding is essential but the nature of the union between tissue and the iron hematoxylin complex was not determined.