Fluorescence microscopy with antisera against specific cellular structure: double photography method for cell identification in populations of multiple cell types

Immunofluorescent staining techniques using antitubulin antibody have been difficult to apply to meiotic tissue (testis) because of the large number of cell types present. Such techniques customarily use a fluorescent dye to counterstain nuclei, and this counterstain is hard to distinguish because of the fluorescence of the antitubulin. By counterstaining with dilute hematoxylin, we can photograph the same field using UV and then conventional illumination. This double photography allows us to identify precisely the many types of cells present, and it will be a useful tool for reexamining the staging of spermatogenesis.     

Synergistic Tissue Counterstaining and Image Segmentation Techniques for Accurate, Quantitative Immunohistochemistry

Quantitative analysis of digitized IHC-stained tissue sections is increasingly used in research studies and clinical practice. Accurate quantification of IHC staining, however, is often complicated by conventional tissue counterstains caused by the color convolution of the IHC chromogen and the counterstain. To overcome this issue, we implemented a new counterstain, Acid Blue 129, which provides homogeneous tissue background staining. Furthermore, we combined this counterstaining technique with a simple, robust, fully automated image segmentation algorithm, which takes advantage of the high degree of color separation between the 3-amino-9-ethyl-carbazole (AEC) chromogen and the Acid Blue 129 counterstain. Rigorous validation of the automated technique against manual segmentation data, using Ki-67 IHC sections from rat C6 glioma and β-amyloid IHC sections from transgenic mice with amyloid precursor protein (APP) mutations, has shown the automated method to produce highly accurate results compared with ground truth estimates based on the manually segmented images. The synergistic combination of the novel tissue counterstaining and image segmentation techniques described in this study will allow for accurate, reproducible, and efficient quantitative IHC studies for a wide range of antibodies and tissues. (J Histochem Cytochem 56:873–880, 2008)     

An autoradioraphic and cytochemical study of erythropoiesis in a fresh water fish, Channa punctatus Bloch

Using spleen and head kidney imprints, studies on erythropoiesis in Channa punctalus have been made describing each developmental stage with regard to its morphology, morpho-metry and cytochemistry. This has been undertaken using the new techniques available for haematopoietic studies of fishes. These include autoradiography for information on DNA synthesis and Graham-Knoll's benzidine method with Giemsa used as a counterstain for the differential staining of haemoglobin. Thus, a more definitive picture of the haematopoietic phenomenon in Channa punctatus has been evolved.     

The Mechanism of the Gram Reaction I. The Specificity of the Primary Dye

Dyes of all major types were tested for their suitability as the primary dye in the Gram stain. When a counterstain was not used, some dyes of all types were found to differentiate Gram-positive from Gram-negative organisms. When a counterstain was used, these dyes were found to vary greatly in their suitability. Those dyes found to be good substitutes for crystal violet were: Brilliant green, malachite green, basic fuchsin, ethyl violet, Hoffmann's violet, methyl violet B, and Victoria blue R. All are basic triphenylmethane dyes. Acid dyes were generally not suitable. Differences in the reaction of Gram-positive and Gram-negative cells to Gram staining without the use of iodine were observed and discussed but a practical differentiation could not be achieved in this manner. Certain broad aspects of the chemical mechanism of dyes in the gram stain are discussed.     

Some fluorescent counterstains for neuroanatomical studies

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.     

Magnifying Stem Cell Lineages: The Stop-EGFP Mouse

Cell fate mapping techniques which can label clonal cell lineages are of importance because they allow one to investigate the distribution and types of daughter cells arising from single precursor cells. Thus, the potential of precursor cells to generate various types of descendent cells can be studied at the single-cell level. The stop-EGFP transgenic mouse carries a premature stop codon-containing enhanced green fluorescent protein (EGFP) gene as a target gene for mutations. A cell having undergone a mutation at the premature stop codon and its descendant cell lineage will express EGFP, thus a clonal cell lineage can be traced in vivo using a fluorescent microscope. Using the stop-EGFP mouse, stem cell clonal lineages in the mouse dorsal epidermis can be investigated in vivo and repeated analyses of the same cell lineages can be performed over time. In vivo imaging studies possible with the stop-EGFP mouse provide new insights into the structure of epidermal proliferative units (EPUs). The stop-EGFP system provides a novel tool for investigating clonal cell lineages in developmental studies as well as in stem cell biology.     

Detailed Kinetic Analysis of Shay Chloroleukaemia Cell Population Propagated In Permanent Suspension Culture In Vitro

Detailed kinetic analysis of a growing cell population is difficult, even when assay conditions are nearly ideal. Therefore, it is usually essential to perform several types of experiments and analyse all the results in terms of a mathematical model, the use of which is not limited a priori by a specified application. In the present study we investigated cell population kinetics using rat chloroleukaemia cells propagated in suspension culture in vitro. the parameters were measured: doubling time of the population, fraction of labelled mitoses, changes in labelling index with time after pulse labelling, continuous labelling and stathmokinetic index. Analysis of the results was based on a computer program CECAM, which is a stochastic model capable of simulating essentially all types of kinetic experiments based on presently known assay techniques. The results showed that precise and reliable information on cell population kinetics could not be obtained from the analysis of any single type of experimental data. In particular, the technique of labelled mitoses underestimated the duration of the G₁ phase, owing to subtle label-induced changes in population behaviour. These changes could not have been detected with any certainty without rigorous quantitative comparisons with other types of experimental data. As a whole, however, results obtained by the different techniques were in agreement and the kinetic behaviour of chloroleukaemia cells in vitro could be established in detail. In certain circumstances even minute changes in the kinetic parameters of the cells can modify population behaviour drastically. to study these cases adequately the experiments must be designed with utmost care, preferably with the aid of preceding simulations. This is because demonstration of small primary changes in population kinetics may be beyond the limit of detection of any single assay method.     

Automated Detection of B Cell and T Cell Acute Lymphoblastic Leukaemia Using Deep Learning

PurposeLeukaemia is diagnosed conventionally by observing the peripheral blood and bone marrow smear using a microscope and with the help of advanced laboratory tests. Image processing-based methods, which are simple, fast, and cheap, can be used to detect and classify leukemic cells by processing and analysing images of microscopic smear. The proposed study aims to classify Acute Lymphoblastic Leukaemia (ALL) by Deep Learning (DL) based techniques.ProceduresThe study used Deep Convolutional Neural Networks (DNNs) to classify ALL according to WHO classification scheme without using any image segmentation and feature extraction that involves intense computations. Images from an online image bank of American Society of Haematology (ASH) were used for the classification.FindingsA classification accuracy of 94.12% is achieved by the study in isolating the B-cell and T-cell ALL images using a pretrained CNN AlexNet as well as LeukNet, a custom-made deep learning network designed by the proposed work. The study also compared the classification performances using three different training algorithms.ConclusionsThe paper detailed the use of DNNs to classify ALL, without using any image segmentation and feature extraction techniques. Classification of ALL into subtypes according to the WHO classification scheme using image processing techniques is not available in literature to the best of the knowledge of the authors. The present study considered the classification of ALL only, and detection of other types of leukemic images can be attempted in future research.     

Propidium iodide as a nuclear counterstain for immunofluorescence studies on cells in culture

We describe a rapid procedure using propidium iodide (PI) as a nuclear counterstain in immunofluorescence studies where cell surface or intracellular antigens are localized with fluorescein-conjugated antisera. In fixed monolayer preparations, all cell nuclei fluoresce red and can be seen simultaneously with cellular antigens that fluoresce green. Counterstaining with PI therefore makes possible quantification of the proportion of cells present in culture that stain immunocytochemically for a specific antigen.     

Demonstrating Weakly Acid-Fast Tubercle Bacteria in Sputum

Acid-fastness of the tubercle bacteria has long been used as the common method of diagnosis in sputum. It has been suggested sometimes that tuberculosis could occur without demonstrable bacteria, as well as with acid-fast bacteria, non-acid-fast bacteria or granules. It is shown in this paper that some of the sputa which are negative to the standard staining technic will show rods, rods with round polar bodies, or similar bodies without the rod portion. It is also pointed out that the decolorization of the smears by acid alcohol be shortened to approximately 3 to 5 seconds and picric acid be used as a counterstain. These forms are apparently the varying stages of the loss of acid-fastness. It is essential that a counterstain be used which will not interfere, and yellow is indicated because it does not absorb the red rays. Sputa which are negative to the standard acid-fast staining technic but which come from persons with a variable intermittent fever should be stained by this modified technic before they are pronounced germ-free.     

Enhancement of Histological Detail Using Metanil Yellow as Counterstain in Periodic Acid Schiff's Hematoxylin Staining of Glycol Methacrylate Tissue Sections

Histological detail in sections from tissues embedded in glycol methacrylate was improved by counterstaining PAS/iron-hematoxylin stained sections with a dilute solution of metanil yellow. The addition of the counterstain increases contrast in tissue sections and highlights PAS-positive entities. The staining protocol provides sharp definition of tissue morphology, differentiates cell types and other tissue components and does not produce background staining.     

The double staining of plant peroxidase and other proteins in the same polyacrylamide gel

We present a simple and rapid technique for the double staining of plant peroxidase and other proteins in the same polyacrylamide gel using the principle of iodide oxidation followed by Coomassie Blue counterstain. The colored bands of peroxidase isozymes and proteins are easily distinguishable. An additional benefit of the method is the use of the low cost chemicals, as well as it eliminates the need for a potentially hazardous reagents frequently used in the detection of peroxidase isozymes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cell behaviour on micropatterned surfaces

The local microenvironment of tissue cells has a profound influence on cell behaviour such as cell shape, guidance of movement, and so on. One approach to understanding this phenomenon, which is being applied by a number of groups, is to model possible cues using microfabrication technology. Such techniques have been used to examine the behaviour of a number of cell types. The responses of fibroblasts, epithelial cells and neurones have been determined on a variety of micropatterned surfaces. Conventional photolithographic techniques and laser holography have been employed to define topographic patterns with feature sizes ranging from 25 μm to 130 nm. Photolithography, combined with silanization of glass, has been used to chemically pattern surfaces; this results in differentially adhesive surfaces that mimic possible in vivo cues. The determination of the response of various cell types to these various surfaces has provided detailed information on the biological mechanisms controlling cell behaviour, and on aspects of tissue responses to implanted artificial devices; it has also illustrated the potential for technology utilizing immobilized cellular patterns.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Applications of DAPI Cytochemistry to Neurobiology

4′, 6-Diamidino-2-phenylindole hydrochloride (DAPI) is a fluorescent dye with high affinity for DNA. We have employed it as a fluorescent chromatin counterstain on sections immunofluorescent-stained using rhodamine and on tissues enzymatically stained using β-galactosidase. DAPI also allows easy identification of mitotic figures and can be used to supplement cytochemical studies involving cell division in the nervous system.     

Improved indirect fluorescence immunocytochemical method using counterstains.

Immunocytochemistry in recent years has provided powerful tools for research in neurobiology. One of the more popular techniques is the indirect fluorescence technique in which fluorescein isothiocyanate (FITC) or tetrahodamine isothiocyanate (TRITC) is used. Although widely used, this technique has two disadvantages: (1) localization may be difficult in relation to background morphology, and (2) the fluorescence fades. The study reported here describes a modification of an indirect immunocytochemical technique using FITC, TRITC and 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) which enhances localization and significantly prolongs fluorescence. Evans blue was used as a counterstain. The results show that FITC and AMCA stained cells are seen against a background of clearly distinguishable cells after counterstaining with Evans blue. However, Evans blue is not compatible with TRITC. In addition, the fluorescence life of the FITC is extended from several days to several weeks with Evans blue. These results clearly indicate that Evans blue can be used to improve indirect fluorescence immunocytochemical techniques.     

The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.     

Staining Iron Bacteria

A modification of Perls's reaction for hemosiderin in tissue is described for staining iron bacteria. The procedure involves the formation of either Prussian blue (ferric ferrocyanide) or Turnbull's blue (ferro ferricyanide) where iron is present on the surface of the bacterium or in the sheath enclosing the bacterium. The counterstain, safranin, imparts a pink color to the noniron-bearing portions of the bacterial cell. Excellent results have been obtained using this technique for staining Clenothrix and Gallionella. The procedure is a valuable aid for the rapid detection of iron bacteria in water samples and for the study of the microscopic morphology and biochemical activities of these organisms.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

A Simplified Method for Staining Mast Cells with Astra Blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however, the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl₂ · 6H₂O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.