A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

Cellular membrane contrast and contrast differentiation with osmium triazole and tetrazole complexes

Summary Addition of heterocyclic nitrogen compounds to the classical osmium tetroxide postfixation medium, applied after glutaraldehyde fixation, results in enhanced membrane contrast in ultrathin sections of liver tissue. The addition of similar compounds to potassium osmate solutions, results in contrast differences in some cellular membranes. The membranes of the rough endoplasmic reticulum, the nuclear envelope and the plasma membrane acquire contrast, while the mitochondrial membranes do not. The apolar regions of membranes are contrasted when osmium tetroxide is combined with heterocyclic nitrogen compounds, whereas the polar regions are contrasted by combinations of potassium osmate with these compounds. This polar membrane contrast is probably due to the presence of an amino-group in the heterocyclic nitrogen compounds. Compounds without the amino-group do not contrast membranes, although the glycogen is contrasted.X-ray microanalysis served to establish the relative osmium content in contrasted glycogen, and showed that such nitrogen compounds play a role in complexation of cations in aldehyde-fixed tissues. Electron spectroscopy for chemical analysis (ESCA) measurements of isolated muscle glycogen show that after treatment with various osmium tetroxide or potassium osmate solutions, hexavalent and quadrivalent osmium species are present in the glycogen. The presence of (heterocyclic) nitrogen compounds in such solutions stabilizes certain osmium valency species, and this may account for the contrast observed.     

Electron staining of the cell surface coat by osmium-low ferrocyanide

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.     

How Romanowsky stains work and why they remain valuable — including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

Abstract

An introduction to the nomenclature and concept of “Romanowsky stains” is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of “the color purple.” Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of “the color purple,” the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a “universal” generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye–basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

Identification of Oleoresin in Epoxy-Embedded Slash Pine Tissue

Sudan black B stains oleoresin blue-black in epoxy-embedded material as well as in living tissue. The Sudan black B staining properties of oleoresin are similar to those of lipid, but it can be distinguished from tannin, which stains brown. Practically all oleoresin present in resin ducts and intercellular spaces, and much of that contained in epithelial and ray cells, is extracted in preparatory procedures for electron microscopy. A fixation procedure is proposed which preserves significantly more oleoresin in situ The use of Sudan black B enables one to localize oleoresin by light microscopy, and permits direct comparison of adjacent sections of epoxy-embedded material at the ultrastructure level. Ultrastructurally oleoresin and lipid possess similar electron densities and can be distinguished from the highly electron-opaque tannin deposits.     

Identification of oleoresin in epoxy-embedded slash pine tissue.

Sudan black B stains oleoresin blue-black in epoxy-embedded material as well as in living tissue. The Sudan black B staining properties of oleoresin are similar to those of lipid, but it can be distinguished from tannin, which stains brown. Pratically all oleoresin present in resin ducts and intercellular spaces, and much of that contained in epithelial and ray cells, is extracted in preparatory procedure for electron microscopy. A fixation procedure is proposed which preserves significantly more oleoresin in situ. The use of Sudan black B enables one to localize oleoresin by light microscopy, and permits direct comparison of adjacent sections of epoxy-embedded material at the ultrastructure level. Ultrastructurally oleoresin and lipid possess similar electron densities and can be distinguished from the highly electron-opaque tannin deposits.     

Silver stains demonstrating neuroendocrine cells

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the “older” silver stains and two of the “newer” ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Effect of some negative stains on the calcium transport of sarcoplasmic reticulum

Summary Effect of some negative stains used in electron microscopy on calcium-transporting membranes of the fragmented sarcoplasmic reticulum (FSR) and interaction between stains and calcium have been studied.Calcium transport as well as ATP-ase activity of FSR are affected by stains investigated at concentrations lower than those used in electron microscopy. Degree of alteration varies from stain to stain. Phosphotungstic acid is the most effective inhibitor.Calcium loaded vesicles are depleted during resuspension in various staining solutions, in concentration routinely used for electron microscopy, according to the calcium affinity of the stain. Also in this respect phosphotungstic acid is the most effective.     

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.     

Iron Hematoxylin Chelates. I. The Weil Staining Bath

The procedure for the preparation of the staining solution for the Weil myelin sheath stain was systematically varied in respect to pH, concentration, time, temperature and relative proportions of the ingredients. The results were explainable on the basis of the presence of a number of iron hematoxylin chelates in the staining bath. Compounds of the form of [Fe_nHem]^m+ are nuclear stains, those of the form of [FeHem_n]^m± are myelin sheath stains while the precipitate is probably [Fe_nHem_y]_x^m±. The following procedure for the stain is recommended. Mix equal portions of a 0.25% solution of ripened hematoxylin prepared from a 10% alcoholic solution and 1% ferric ammonium sulphate and use immediately. Preferably, the solutions should be at a temperature of about 5 C and the staining done in the refrigerator, but room temperature may be used. Higher temperatures are contraindicated. Hematein should not be substituted for ripened hematoxylin; the resulting stains are too weak to be usable. The absorbance of hematein is no measure of the concentration of the component that stains myelin sheaths. Hematein apparently consists largely of a sparingly soluble highly colored inactive compound.     

Ultrastructure and staining properties of aortic microfibrils (oxytalan)

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

Functional and structural analysis of acetylcholine receptor-rich membranes after negative staining

Phosphotungstate (pH 7.4) used for negative staining of membranes from Torpedo electric tissue rich in acetylcholine receptor does not affect binding properties and cation permeability of the receptor and its ion channel. Uranyl salts, frequently used for negative staining, precipitate the receptor-rich membranes making measurements of ligand binding and ion-permeability regulation impossible. The gross ultrastructure in the two stains is not significantly different, but for future high-resolution electron microscopy aiming at visualizing structural details of functional receptor molecules it is necessary to resort to a stain preserving native and active receptor. Uranyl salts are not applicable for this purpose. The electron micrographs obtained with phosphotungstate reveal two distinct structures in the receptor-rich membrane: a closed ring ('doughnut') and an open ring ('horseshoe'), with a ratio of abundance of about 3:2.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

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.     

Osmium-labeled polysaccharides for atomic microscopy

With the objective of localizing cell surface polysaccharides, the reaction of several osmium (VI)-ligand complexes with glycols has been applied to sugar residues in mono- and polysaccharides. The hydrophilic ligands 4,4′-dicarboxy-2,2′-bipyridine and N,N,N′,N′-tetramethylethylenediamine have been employed to produce water-soluble osmate esters of the sugar glycols. Methyl glycosides react with osmium (VI) reagents to give stable products containing one osmium atom per sugar. α-Cyclodextrins, with six glucose residues in a ring, and β-cyclodextrins with seven, can bind up to, but not exceeding three osmium ligand complexes per molecule, indicating possible nearest-neighbor exclusion of reaction among the residues. Reaction of as little as 1% of the sugar residues in amylose with the dicarboxybipyridyl osmate complex allows the amylose strands to be extended on a grid for electron microscopy. Further reaction with the tetramethylethylenediamine osmate complex rapidly saturates the amylose to a level of 0.39 osmium atom per sugar residue, consistent with the nearest-neighbor exclusion hypothesis. High resolution scanning transmission electron microscopy images reveal a row of osmium atoms along each amylose strand.     

Preservation of alveolar type II pneumocyte lamellar bodies for electron microscopic studies.

Simultaneous fixation with glutaraldehyde and osmium tetroxide, followed by an uranyl acetate (UA) treatment before dehydration and embedding (Hirsch and Fedorko 1968) ensures a very good preservation of lamellar bodies (LB's) as well as of the cellular membranes in type II pneumocyte. The uranyl acetate treatment appeared to be the most efficient step of the procedure. The morphological aspect of lamellar bodies after such a preparation was similar to that observed after freeze-etching of lipid retaining methods. Moreover, the Hirsch-Fedorko procedure is very simple and can easily be used for routine ultrastructural and radioautographic studies. On the other hand, it appeared that the uranyl acetate phospholipid "complex" is very sensitive to the pH of chemical solutions used after sectioning. The "complex" is variously dissolved by alkaline solutions, photographic developers or stains. The best preservation of ultrastructure was obtained with neutral or acidic developers and acidic stains.