A Controllable Differential Stain for the Hypophysis by Adaptation of the Mallory Triple Stain

A selective and controllable staining method for the hypophysis has been developed with rat material, using Mallory's triple stain as a basis.

Fix in Zenker neutral formol for 6 hours. Longer fixation is undesirable. Transfer to 30% alcohol plus a few drops of a saturated solution of I₂ in aqueous KI over night. Gradually complete dehydration and clear in cedar oil. Infiltrate with a paraffin mixture (paraffin, rubber-paraffin, bayberry wax and beeswax). Section 3-Sμ. Hydrate to distilled water, placing a few drops of a KI-I₂ solution in the 50% alcohol. Stain in 1% acid fuchsia for 30 minutes. Rinse, and differentiate in a weak NH₄OH solution (one drop 28% NH₄OH to 200 cc. HOH). When differentiation is complete, transfer to a 0.5% phosphomolybdic acid solution for 3 minutes, after first stopping the differentiation with a 0.1% HC1 solution and then rinsing with distilled water. Stain for one hour in a solution of: 1% anilin blue, water soluble, 2% orange 6, and 1% phosphomolybdic acid. Rinse in distilled water plus a few cubic centimeters of the stain. Differentiate in 95% alcohol, transfer to absolute alcohol and clear in a mixture of 30% oil of cedar, 40% oil of thyme, 15% absolute alcohol and 15% xylene. Finally, transfer to xylene and mount.     

An Azocarmine stain for Differential cell Analysis of the rat Anterior Hypophysis

A method of staining is described which is especially designed to facilitate differentiation of the cell types of the rat anterior hypophysis. Fixation in Zenker-formol solution is recommended. Pre-staining of the nuclei by a short immersion in alum hematoxylin is followed by mordanting in anilin alcohol and a 45 minute period of staining in azocarmine solution at 60d`C. The counterstains, acid green and orange G, are dissolved in clove oil to avoid destaining of the azocarmine.     

Congo Red as a Simple Stain for Beta Cells of the Hypophysis

Bennhold's Congo red method for amyloid has been found to provide a simple, one step, differential staining technique for cells of the anterior lobe of the hypophysics, and to be applicable to routine use with formalin-fixed tissue. The beta cells stain orange-red and the alpha cells yellow, while the chromophobe cytoplasm remains unstained. This method can be used on material from humans and laboratory animals. Ordinary degrees of post-mortem change do not affect the staining reaction.     

A Staining Method for the Anterior Hypophysis of the Rat

A staining procedure for the anterior hypophysis of the rat, differentiating between eosinophilic granules, basophilic granules and mitochondria, has been divised. Small pieces of hypophyseal tissue are fixed in Champy's fluid. Following fixation the tissue is either chromated or osmicated. After being embedded in 60-62° paraffin, the tissue is cut serially at 2 and 3 μ. The sections are stained with 7% Altmann's acid fuchsin by heating on a laboratory hot plate, followed by 30 seconds in a 2% solution of Orange G made up in 1% phosphomolybdic acid. They are then treated for 10 seconds in a .01% solution of potassium carbonate, and stained for 10-30 minutes in Goodpasture's acid polychrome methylene blue. The mitochondria stain brilliant fuchsia, the eosinophilic granules orange-red, and the basophilic granules deep blue.     

Congo Red as a Simple Stain for Beta Cells of the Hypophysis

Bennhold's Congo red method for amyloid has been found to provide a simple, one step, differential staining technique for cells of the anterior lobe of the hypophysics, and to be applicable to routine use with formalin-fixed tissue. The beta cells stain orange-red and the alpha cells yellow, while the chromophobe cytoplasm remains unstained. This method can be used on material from humans and laboratory animals. Ordinary degrees of post-mortem change do not affect the staining reaction.     

Gross Differential Staining of the Hypophysis

The lobes of the hypophysis of many mammals can be differentiated by staining either the entire gland for 5–8 hr, or gross 1–2 mm slices of the gland for 3–5 min in a mixture of acid fuchsin (National Aniline, certified Andrade indicator) and methylene blue (Fisher certified reagent). For best results, the staining mixture contains 0.8–1.0% acid fuchsin and 0.2–0.4% methylene blue, both made up in 10% formalin adjusted to pH 6.70–6.75 with phosphate buffer. The anterior lobe stains a light blue, the intermediate lobe a dark blue, the posterior lobe a light pink and the capsule a dark pink.     

Electrophysiology of the Human Hypophysis

Electrical activity was recorded in the human hypophysis from the neurosecretory fibres of the posterior lobe. In seven patients undergoing trans-sphenoidal hypophysectomy, electrophysiological recording was carried out with a special fine-shielded bipolar concentric electrode with a tip diameter of 30 microns. The rate of unit impulses was modified by anesthetic agents and after intravenous injections of drugs acting at the hypothalamic level.This technique was used to detect the limit between the anterior and posterior lobe in order to perform a selective adeno-hypophysectomy by implantation of yttrium-90 seeds.     

A Differential Stain for Nucleic Acids

An easily used trichrome stain consisting of orange G, methyl green, and toluidine blue is proposed as a method of differentiating desoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in cells. Carnoy's acetic-alcohol is the fixative of choice, though cold acetone is also satisfactory. Photomicrographs taken with ultraviolet and visible light show that the structures containing nucleic acid are exactly those which stain with methyl green and toluidine blue. Studies with nucleases and extraction of nucleic acids with cold and hot perchloric acid further indicate a specificityy of the dyes for DNA and RNA. Present experiments are directed toward using the stain for quantitative estimation of the nucleic acids.     

Differential Stain for acid fast Bacteria and Spores

A modified method of staining acid-fast organisms is described. After staining with carbol-fuchsin as usual in the Ziehl-Neelson method, wash with water and while the slide is still wet cover with a saturated acetone solution of malachite green for three to five minutes. Wash and examine. The acid-fast organisms and spores are red in a green background. If the smear is thick and appears too dense, dry for three minutes and hold over the mouth of a bottle of ammonia until decolorized to suit. Upon exposure to the air the green returns. This can be prevented by keeping the smear alkaline, by the addition of sodium bicarbonate.

A second method is described for use with sputum in which acid-fast organisms are scarce. It permits the examination of thick smears and therefore increases the chances of finding tubercle organisms when few in number. Stain with carbol fuchsin as in the Ziehl-Neelson method. Decolorize with 30% phenol-disulfonic acid in water for a few seconds or until decolorized. Wash and examine at once. If color returns upon washing decolorize again. The tubercle organisms appear red in a colorless background.     

A Differential Stain for Rubber in Guayule

The following schedule, which combines an intense blue stain for rubber with sharply contrasting red counterstains, has been found satisfactory for use in an anatomical study of rubber deposition in guayule: Cut fresh or fixed sections about 50 to 100 % thick, transfer to 50% ethanol. Extract with acetone 5 minutes, treat with 1% NaOCl 5 minutes, saponify with 10% KOH in 95% ethanol 15 minutes, rinse 3 times with 50% ethanol, stain in oil blue NA (Calco) with safranin and Congo red 30 minutes at 55° C. Rinse in 50% ethanol 2 (or more) times to remove excess stain and mount in Karo syrup.     

Simultaneous Specific Demonstration of Thyrotroph, Gonadotroph and Acidophil Cells in the Anterior Hypophysis

A staining sequence for the simultaneous demonstration of thyrotrophs, gonadotrophs, and acidophils in the anterior hypophysis is described. It consists of permanganate oxidation, aldehyde-thionin, luxol fast blue, periodate oxidation and leuco fuchsin. By this technique rat and dog pituitaries fixed in mercuric chloride-formalin show blue-black thyrotrophs, red gonadotrophs, green acidophils and unstained or faintly stained chromophobes.     

Wright's as A Differential Spore Stain

A method of differential spore staining utilizing Wright's stain diluted one to five in a phosphate buffer solution of pH 7.6 and following the general technic of the Dorner method is outlined. Spores are stained a deep blue while the cytoplasm of the sporangium is stained a pinkish red.     

A Hypochlorite-Fast Metachromatic Quick Stain for Human Megakaryocytes

Megakaryocytes in aspirates of normal and abnormal human bone marrow displayed rose-pink metachromasla when stained with a violet polymethine dye. The color of the megakaryocytes persisted when the stained sample was exposed to a dilute solution of sodium hypochlorite. Under the same conditions, other hematopoietic cells became colorless. This stain may be useful for demonstrating megakaryocyte from both healthy and diseased bone marrow specimens.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

Wright's as A Differential Spore Stain

A method of differential spore staining utilizing Wright's stain diluted one to five in a phosphate buffer solution of pH 7.6 and following the general technic of the Dorner method is outlined. Spores are stained a deep blue while the cytoplasm of the sporangium is stained a pinkish red.     

Oil Blue N or Na as a Fat Stain for Animal Histology

Oil blue N or NA gives precise dark blue staining of fatty substances in animal tissues when supersaturated solutions in dilute isopropanol are used. A stock saturated solution in 60% isopropanol, when diluted to 40 or 50% isopropanol, gives good staining in 5 to 10 minutes, without appreciable precipitate.

Suitable counterstains are 1:1000 solutions of Janus green B, Bismarck brown Y and Bismarck brown R. These require about 5 minutes and should be followed by one-minute differentiation in 5% acetic acid. Of the three, Bismarck brown R gives the best contrast.     

Cytokines in the Hypophysis: A Comparative Look at Interleukin-6 in the Porcine Anterior Pituitary Gland

Interleukin-6 (IL-6) is a multifunctional cytokine produced by a variety of cell types in tissues of both the immune and endocrine systems. Among the major functions described for IL-6 are its role in the maturation of B cells to high-output antibody-producing cells and its contribution to the acute physiological responses to infection and inflammation, notably production of hepatic acute phase proteins and activation of the hypothalamic-pituitary-adrenal axis. In addition to these better known functions, IL-6 recently has been found within the pituitary of laboratory rats and also in the human pituitary. In rats, pituitary IL-6 mRNA is upregulated by peripheral exposure to bacterial endotoxin. However, the role of anterior pituitary IL-6 in host responses to infection and inflammation remains uncertain, although it may regulate pituitary hormone secretion. The following brief review summarizes the information available concerning cytokine production within the anterior pituitary of species of domestic livestock. To our knowledge, experiments conducted in our laboratory evaluating regulation of IL-6 mRNA expression and secretion from the porcine anterior pituitary provide most of the data in domestic species confirming the presence of IL-6 in the pituitary. Our data indicate that IL-6 mRNA is present in cultured porcine anterior pituitary cells and that the pituitary directly responds to stimulation with bacterial endotoxin by increasing secretion of IL-6. Furthermore, endotoxin-induced upregulation of IL-6 mRNA expression and secretion appears to be dependent upon production of cyclooxygenase products of arachidonic acid metabolism.     

A Differential Stain Favorable to the Diagnosis of Neisserian Infection

A differential Gram stain has been evolved which incorporates the combined features of the original Gram and Pappenheim methods. National Aniline crystal violet and new methyl green and pyronin are the dyes preferred. The iodine mixture of Kopeloff and Beerman is a satisfactory mordant and Merck's pure technical acetone is an excellent differentiating agent. A system is established by means of the dyes and reagents which form a physicochemical equilibrium, provided pure dyes are employed, and the technic is carried out with precision. Gram-positive bacteria are coated by means of buffered crystal violet solution and the iodine-sodium hydroxide solution precipitates the crystal violet from other substances. The dye-iodine precipitate is readily dissolved by pure acetone. Iodine green, a pure derivative of crystal violet has the effect of noninterference in the technic and has selective action upon nuclear substance. Pyronin has affinity for Neisserian organisms primarily and acts as an inert substance upon most other proteins, (except cytoplasm of eosinophils, lymphocytes, plasma cells, and endothelial cells). The following technic is recommended:

Stain air-dry films 3 to 5 minutes in a 1% solution of crystal violet in 10 parts of Clark and Lubs' phosphate buffer of pH 6.6 to 7.0 and 90 parts water. Decant and flush with 2% iodine in N/10 NaOH. Decant and decolorize in acetone 10 seconds or less. Air dry and counterstain 1 1/2 to 2 minutes with methyl-green-pyronin (2 parts 2% aqueous methyl green National with one part 0.3% aqueous pyronin yellowish). Wash and air dry. Oil of Bergamot is preferable to xylene as a clearing agent. Best results are obtained if each slide is handled separately as for staining blood films.