Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

Uniform Staining of Nerve Endings in Skeletal Muscle with Gold Chloride

Skeletal muscle, teased into strips several millimeters long and 1 mm or less thick, is fixed in a 3:1 mixture of lemon juice and formic acid until transparent. The tissue is compressed between folds of absorbent material to remove excess fixative and impregnated for 10 min in a volume of 1% gold chloride equal to the volume of muscle. After removing excess gold chloride, reduction is effected. Method a. Exposure to 25% formic acid in the dark for 6 hr. When intramuscular nerves are visible, the muscle is further teased with glass instruments. Reduction is continued for an additional 18 hr in 25% formic acid, and the tissue is cleared in glycerol for 24 hr. Method b. Exposure to intense artificial visible light while the tissue is just immersed in distilled water at 37° C. Colour develops in the nerve endings as in a photographic print. When nerves only are stained (approximately 0.5 hr), the lamp is removed and microdissection at room temperature commenced. When nerve endings in muscle spindles are just visible (approximately 1 hr), the distilled water is replaced by glycerol. After either method of reduction, areas of motor end-plates and individual sensory receptors are isolated by microdissection. Preparations are mounted in a small drop of glycerol, considerable pressure is applied to the cover slip, and the cover ringed with a sealing fluid.     

Diagnostic Value of Immunohistochemical Staining of GP73, GPC3, DCP,CD34, CD31, and Reticulin Staining in Hepatocellular Carcinoma

It has been reported that Golgi protein-73 (GP73), glypican-3 (GPC3), and des-γ-carboxy prothrombin (DCP) could serve as serum markers for the early detection of hepatocellular carcinoma (HCC). This study aimed to evaluate a panel of immunostaining markers (including GP73, GPC3, DCP, CD34, and CD31) as well as reticulin staining to distinguish HCC from the mimickers. Our results revealed that CD34 immunostaining and reticulin staining were highly sensitive for the diagnosis of HCC. A special immunoreaction pattern of GP73—a diffuse coarse-block pattern in a perinuclear region or a concentrated cluster-like or cord-like pattern in a certain part of the cytoplasm—was observed in HCC cells, in contrast to the cytoplasmic fine-granular pattern in surrounding non-tumor cells and non-malignant nodules. This coarse-block pattern correlated significantly with less differentiated HCC. In comparison, GPC3 displayed a good advantage in diagnosing well-differentiated HCC. In our study, DCP and CD31 showed little diagnostic value for HCC as an immunostaining marker. When GP73, GPC3, and CD34 were combined, the specificity improved to 96.6%. Our findings demonstrate for the first time that the immunohistochemical panel of GP73, GPC3, and CD34 as well as reticulin staining is highly specific for the pathological diagnosis of HCC.     

Staining with Phloxine

A double stain with Magdala red and anilin blue has sometimes given very satisfactory results; but, just as often, has been entirely worthless. The reason for the discrepancy seems to be that stains sold under the name of Magdala red are of various composition, some of them containing no Magdala red at all. The standardized stain phloxine seems to be identical with successful lots of Magdala red and results are rather uniformly successful. Detailed directions for staining with phloxine and anilin blue will be published in a forthcoming number of Stain Technology.     

Staining with Phloxine

A double stain with Magdala red and anilin blue has sometimes given very satisfactory results; but, just as often, has been entirely worthless. The reason for the discrepancy seems to be that stains sold under the name of Magdala red are of various composition, some of them containing no Magdala red at all. The standardized stain phloxine seems to be identical with successful lots of Magdala red and results are rather uniformly successful. Detailed directions for staining with phloxine and anilin blue will be published in a forthcoming number of Stain Technology.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Improved Gold Chloride Procedure for Nerve Staining in Whole Mounts of Rat Corneas

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryo-protective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at-70 C in O. C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO₄-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO₄-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO₄-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O. C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced nonspecific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohisto-chemical procedures where the reaction products would be obscured by background staining.     

Differential Nucleolar Staining Affinity with a Modified Papanicolaou Staining Procedure

A modified Papanicolaou staining procedure using diluted Harris' hematoxylin with potassium alum is described. Nucleolar staining varies from blue to bright red. This technique has been applied to mammary tumor cell lines in vitro under several conditions of hormonal stimulation known to induce protein synthesis and cell differentiation. Blue nucleoli are observed in control resting cells, while bright red nucleoli are seen after hormonal stimulation.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30-90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Histological Staining with Methyl-Green-Pyronin

The standard technics for methyl green-pyronin staining are found to give inconstant results, often with poor differentiation between chromatin and cytoplasm. A modified procedure is described using n butyl alcohol for differentiation after aqueous methyl green staining and counter-staining with pyronin in acetone. After 6 minutes in 0.2% aqueous methyl green (chloroform extracted), the section is blotted, differentiated in n butanol, counter-stained 30–90 seconds in acetone saturated with pyronin (less concentrated solutions may be preferred for some purposes), cleared in cedar oil and xylene and mounted. This technic retains the value of methyl green as a histochemical detector for polymerized desoxyribo-nucleic acid (DNA). The intensity of the stain, however, is considerably greater than that obtained with the procedure designed for quantitative (stoichiometric) photometric estimation of polymerized DNA. Pyronin serves primarily as a counterstain, and is not found to be a reliable indicator of ribonucleic acid either by this method or others which have been described.     

Staining of Nerve Fibers with Methylene Blue Factors Improving the Staining

Several factors influencing the staining of nerve fibers with methylene blue, especially the influence of chloralhydrate and carbamylcholine chloride (as parasympathicotonics), and of some anesthetics were studied. The intestines of mouse, rat, and guinea pig were used. The following immersion technic is suggested: Tissue from animals anesthetised by chloralhydrate is immersed in: zinc free methylene blue, 0.03%; sodium tartrate, 0.5%; sodium pyruvate, 0.05% carbamylcholine, 0.00005%; 0.2 M Na₂HPO₄, 0.77%; 0.1 M citric acid, 0.18%; NACl, 0.79%; also an anesthetic which varies with the animal selected. Air is kept bubbling through the staining solution and microscopic examination is made at 6 min. intervals. After 0.5-1 hr. the tissue is fixed in: ammonium molyb-date, 10 g.; sucrose, 35 g.; distilled water, 100 ml.; to which is added just before use, 1% platinum chloride, 3 ml.; 2% osmic acid, 3 drops. Washing is in ice cold water and dehydration at 0°C. in Lang's fluids (varying mixtures of ethanol and n-butanol). The tissues thus prepared are stored in liquid paraffin.     

Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

Staining with Chromoxane Cyanine R

Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needs. (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H₂SO₄ (v/v); boil this solution for 5 min, (2) 10% FeCl₃ in 3% HCI, (3) 1% aqueous NH₄OH, and (4) 1% HCI in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl₃ solution, 8 ml H₂O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and win. Histochemical tee show that the functional pup for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.     

Staining Root-Tip Smears with Aceto-Carmine

A number of schedules have been suggested for staining root-tip smears with aceto-carmine (Brown, 1937; Burrell, 1939; Ganeshaw, 1939; Howe, 1946; Smith, 1947; Warmke, 1935). The variation that exists between plant species has undoubtedly been responsible for many of these modifications. In addition, the nature of the information desired has also been a determining factor in the development of new technics (Aisima, 1941; Howe, 1943).     

Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

Permanent Staining with Iodine Vapor

Sections of cooked rice have been stained with iodine vapor, and the method has given greater permanency than any heretofore reported. The method, which permits the use of a counter-stain, has been applied also to starches, starch pastes, uncooked rice flour, and cooked rice flour. The resulting color is of moderate intensity, so that structure is revealed and the birefringence of ungelatinized starch is not impaired. Preparations made more than a year ago have retained their characteristic blue or lavender color.