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.     

Staining Glycol Methacrylate Embedded Cartilage with Triethyl-Carbocyanin Dbtc (“Ethyl-Stains All”) With Special Reference to the Interlacunar Network

The dye, triethyl-carbocyanin DBTC, was tested for differential staining of cartilage structures. Femoral head articular cartilage from neonatal rats was processed for histology to demonstrate the interlacunar network. Sections of glycol methacrylate (GMA) embedded cartilage were stained at pH 2.8, 5.4, 6.1 and 8.0 to determine the optimal staining conditions. Only at pH 6.1 were all cartilage structures stained and the best contrast achieved. Streptomyces hyaluronidase, chondroitinase ABC, pepsin, trypsin, and pronase digestions were carried out prior to staining at pH 6.1 to evaluate the selectivity of the stain. Undigested chondrocyte nuclear chromatin stained dark purple; staining intensity was reduced slightly by pepsin or trypsin digestion. Undigested chondrocyte cytoplasm stained light blue but stained purple after hyaluronidase digestion. Undigested extracellular matrix stained light violet; staining was almost entirely eliminated by chondroitinase ABC digestion, was unaffected by hyaluronidase, and was either unaffected or increased after proteinase digestion. Staining of a narrow zone of matrix adjacent to the network was prevented by proteinase digestion while the network element appeared as a thin dark line. The network appears to be a trilaminar structure; a core element of hyaluronic acid and protein surrounded by a protein sheath. Triethyl-carbocyanin DBTC staining of cartilage offers slightly more selectivity and contrast than methylene blue, toluidine blue or safranin O. At pH 6.1, DNA, perhaps RNA, and hyaluronic acid stained deep purple; chondroitin sulfate, light violet; protein (collagen), stained very light violet if at all.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

A Suction Device for Easy Handling of Glass Coverslips

Electron microscopic data on methylene blue staining of dendritic cells in the epithelia of the soft palate and skin of the moose after supravital dye injection are presented. The ultrastructural details were compared with corresponding light microscopic findings. Methylene blue stained tissue was fixed by immersion in a paraformaldehyde-glutaraldehyde solution containing phosphomolybdic acid. The ensuing dye precipitate was stabilized by ammonium heptamolybdate. The light microscopic investigation revealed that selective staining of dendritic cells depended on the presence of ambient oxygen. In addition, delicate morphological characteristics, like spinous structures of the dendrites, were visible. Some cells also showed terminal enlargements of the dendrites close to the surface of the epithelium. In general, visualization of morphological detail was superior to that obtained by conventional histological and immunohistochemlcal procedures. Nerve fibers were also stained within the epithelium as well as the subepithelial connective tissue. At the electron microscopic level, the dye was clearly identified as an electron dense precipitate that accumulated primarily within the cytoplasm near the plasma membrane. Furthermore, it was bound to the chromatin of the nuclei. No significant staining of mitochondria or other organelles was seen, within the cytoplasm, the oxygen-dependent binding sites may be associated with heme proteins that attract both the dye in its reduced lipophilic leuco form and oxygen, followed by generation of oxygen radicals and a reoxldation of the leuco form to the cationic blue dye. Because of its selectivity for intraepithelial dendritic cells, the method described here supplements immunocytochemical procedures at both the light and electron microscopic levels.     

Supravital Uptake of Methylene Blue by Dendritic Cells within Stratified Squamous Epithelia: a Light and Electron Microscope Study

Electron microscopic data on methylene blue staining of dendritic cells in the epithelia of the soft palate and skin of the moose after supravital dye injection are presented. The ultrastructural details were compared with corresponding light microscopic findings. Methylene blue stained tissue was fixed by immersion in a paraformaldehyde-glutaraldehyde solution containing phosphomolybdic acid. The ensuing dye precipitate was stabilized by ammonium heptamolybdate. The light microscopic investigation revealed that selective staining of dendritic cells depended on the presence of ambient oxygen. In addition, delicate morphological characteristics, like spinous structures of the dendrites, were visible. Some cells also showed terminal enlargements of the dendrites close to the surface of the epithelium. In general, visualization of morphological detail was superior to that obtained by conventional histological and immunohistochemlcal procedures. Nerve fibers were also stained within the epithelium as well as the subepithelial connective tissue. At the electron microscopic level, the dye was clearly identified as an electron dense precipitate that accumulated primarily within the cytoplasm near the plasma membrane. Furthermore, it was bound to the chromatin of the nuclei. No significant staining of mitochondria or other organelles was seen, within the cytoplasm, the oxygen-dependent binding sites may be associated with heme proteins that attract both the dye in its reduced lipophilic leuco form and oxygen, followed by generation of oxygen radicals and a reoxldation of the leuco form to the cationic blue dye. Because of its selectivity for intraepithelial dendritic cells, the method described here supplements immunocytochemical procedures at both the light and electron microscopic levels.     

Thiazin Dyes in Supravital Staining of nerve Fibers

Supravital staining by thiazins of segments of small intestine and mesentery of young dogs was studied with reference to specificity for nervous tissue. Attempts to secure a purer form of methylene blue by alumina adsorption and alcohol elution of the commercial, medicinal dye yielded a product which appeared to be structurally different from the original dye. The treated dye had absorption maxima from 620 to 655 mμ in contrast with 665 for the untreated. Small nerve bundles were stained by the treated dye after 2 to 4 hours of immersion, but staining was always incomplete. Staining by untreated methylene blue was compared with that by the leucobase, thionol, methylene green, toluidine blue, new methylene blue and the azures. It was concluded that the specificity for nerve fibers resides mainly in the =N(CH₃)₂Cl radical, although some specificity appears to be effected by the methyl groups on the trivalent nitrogen, since azure A (dimethyl) and azure C (mono-methyl) stained weakly, but thionin did not. Methylene green showed some specificity but stained weakly. The leucobase was less active than the reoxidized dye obtained from it.     

Romanowsky-type staining: Enzymatic analysis of nuclear metachromasia in formalin-fixed paraffin sections

The red color of nuclei produced in formol-fixed paraffin sections stained with toluidine blue has been investigated by using deoxyribonuclease (DNase), ribonuclease (RNase) and 0.1 M Tris buffer. The action of DNase on formol-fixed material is not fully reliable, but clear-cut when positive. Nuclear basophilia and metachromasia is removed, nucleolar and cytoplasmic RNA is preserved. The picture produced by RNase depends to some extent on the concentration and acidity of the toluidine blue used for subsequent staining. Cytoplasmic RNA is always removed, while the red stain in nuclei usually remains intact. With 0.1% toluidine blue in 1% acetic acid, a nuclear color change from red to pale green is observed. Using this same staining solution, it can be shown that 0.1 M Tris buffer (overnight extraction at 37° C) will remove cytoplasmic RNA but leave intact the nuclear material that stains red. A red to green shift can subsequently be produced by RNase. From this it is deduced that there is a chromatin-associated nuclear RNA fraction which can be removed by the enzyme, but is stable to the buffer solution.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Differential dichrome staining of tissue culture monolayers: alternate dyes and possible mechanism.

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Differential Dichrome Staining of Tissue Culture Monolayers: Alternate Dyes and Possible Mechanism

A polyacid-dependent dichrome has been devised which will differentiate epithelial from mesenchymal cells in young dividing primary cultures. Epithelial cells and colonies and nuclei are stained with metanil yellow, the stain is fixed and differentiated with phosphotungstic acid, and the mesenchymal elements are stained with toluidine blue. Several other dyes are tested for substitution in this method. Biebrich scarlet and aniline blue could be substituted for the metanil yellow; Bismarck brown T, Janus green B, crystal violet, and neutral red could be substituted for the basic dye.     

Early differentiation in zebra fish blastula: Role of yolk syncytial layer

Summary The blastomeres of the zebra fish embryo can be classified into two types-cells stained densely (D) or lightly (L) with a mixture of toluidine and methylene (T-M) blue. The dense staining of D cells is largely due to the high density of mitochondria, rough endoplasmic reticulum and polyribosomes. The presence of partially dense stained cells during early blastula stage shows that L cells are transformed into D cells. That the yolk syncytial layer (YSL) plays some role in this transformation is suggested by the proximity of these cells to the YSL and by their distinct spatial orientation with densely stained cytoplasmic regions always facing towards the interior of the embryo.     

The use of a basic dye (azure A or toluidine blue) plus a cationic surfactant for selective staining of RNA: a technical and mechanistic study.

Selective purple staining of RNA-rich structures such as basophilic cytoplasms of exocrine pancreas and plasma cells, Nissl substance, and nucleoli was achieved by treating tissue sections as follows. Stain dewaxed sections for 1/2 hour in a dyebath containing 0.1% w/v axure A or toluidine blue and 1% cationic surfactant (Hyamine 2389, a 50% w/v aqueous solution of diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride; or benzyldimethylammonium chloride, or cetylpyridinium bromide, or cetyltrimethylammonium bromide) buffered to pH 7 with phosphate. Rinse in water, blot, air dry and mount in synthetic resin. Intense purple staining of RNA-rich regions occurred after fixation in neutral formalin or in Carnoy's or Gendre's fluids, though satisfactory results were also found after fixation in acetone or alcohol. Chromatin generally stained a very pale azure after all fixations, though occasionally nuclei were unstained (Gendre's or Zenker's fluids). Subjecting tissue sections to acid hydrolysis or to digestion by RNAase eliminated or reduced the purple staining, but left the azure staining of nuclei unaffected. Satisfactory staining of RNA-rich structures was not critically dependent on the precise concentrations of dye, surfactant or inorganic salts in the dyebath, nor on pH, staining time or chemical nature of the surfactant. The staining patterns can be rationalized with a tissue model that considers both surface charge and permeability factors, since present in the dyebath are small dye cations and large cationic surfactant micelles. As micelles and dye will both quickly penetrate basophilic structures considered to be porous, such as chromatin, competition will then greatly reduce staining of such substrates. But the large micelles will only slowly penetrate regions considered to be more impermeable, such as basophilic cytoplasms, so consequently small fast moving dye ions may enter and stain without competition.     

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

Summary It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10^–3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new method for identification of heterocysts and proheterocysts in morphologically complex cyanobacteria

A new light microscopic method for identifying heterocysts and proheterocysts in morphologically complex cyanobacteria was evaluated for reliability and usefulness. Mature heterocysts and proheterocysts could be distinguished readily from vegetative cells in 0.25 micron sections of fixed and embedded material after staining with toluidine blue. Examination by light and electron microscopy of the same specimens indicated that the staining reactions which served to differentiate these cell types were both reproducible and accurate. Light microscopic analysis of serial sections stained with toluidine blue greatly facilitated localization of heterocysts and proheterocysts in the complex, branching cyanobacterium, Mastigocladus laminosus, even when its filaments of cells were intertwined in thick mats.