An improved method for clearing and staining free-hand sections and whole-mount samples

BACKGROUND AND AIMS: Free-hand sectioning of living plant tissues allows fast microscopic observation of internal structures. The aim of this study was to improve the quality of preparations from roots with suberized cell walls. A whole-mount procedure that enables visualization of exo- and endodermal cells along the root axis was also established. METHODS: Free-hand sections were cleared with lactic acid saturated with chloral hydrate, and observed with or without post-staining in toluidine blue O or aniline blue. Both white light and UV light were used for observation. Lactic acid was also used as a solvent for berberine, and fluorol yellow for clearing and staining the samples used for suberin observation. This procedure was also applied to whole-mount roots with suberized celllayers. KEY RESULTS: Clearing of sections results in good image quality to observe the tissue structure and cell walls compared with non-cleared sections. The use of lactic acid as a solvent for fluorol yellow proved superior to previously used solvents such as polyethylene glycol-glycerol. Clearing and fluorescence staining of thin roots such as those of Arabidopsis thaliana were successful for suberin visualization in endodermal cells within whole-mount roots. For thicker roots, such as those of maize, sorghum or tea, this procedure could be used for visualizing the exodermis in a longitudinal view. Clearing and staining of peeled maize root segments enabled observation of endodermal cell walls. CONCLUSIONS: The clearing procedure using lactic acid improves the quality of images from free-hand sections and clearings. This method enhances the study of plant root anatomy, in particular the histological development and changes of cell walls, when used in combination with fluorescence microscopy.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques.

Feulgen nuclear staining with pararosanilin-SO2 was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell. The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality between the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by B?hm et al. (1968) and a reaction with ninhydrin at 37 degrees C for 10 h. The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure. Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Summary Feulgen nuclear staining with pararosanilin-SO₂ was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell.The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality beween the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37° C for 10 h.The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure.Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.Partly supported by Deutsche Forschungsgemeinschaft (DFG), grant Nr. Bo 395/4     

Lactic Acid Clearing and Fluorescent Staining for Demonstration of Sieve Tubes

Sieve tubes of the phloem in cleared plant parts can be located by means of a staining reaction specific for callose. The plant part is decolourized in 1:3 glacial acetic acid-95% ethanol and cleared in hot 85% lactic acid at 98-100 C. Callose is not dissolved by this treatment and is then stained with 0.01% analine blue in 0.07 M phosphate buffer, pH 7.5, and observed by fluorescence microscopy. A sieve tube is recognized by the bright yellow fluorescence of the callose on its sieve plates. In most tissues, a natural light yellow fluorescence of the parenchyma cells is evident after the clearing step. This intensifies upon staining with analine blue and tends to make the tissue opaque, but it can be minimized by quick-killing of the tissue before commencing the decolourization. The procedure gives best results when applied to young tissues in which interference from the natural yellow fluorescence of lignified cells such as xylem elements and phloem fibers is minimal. Callose plugs in pollen tubes were also shown in intact, cleared styles.     

Studies on Textile Dyes for Biological Staining II. A Trichrome Stain for General Use

This trichrome staining procedure differentially stains elastic fibers, collagen fibers and mucin. Gomori's aldehyde-fuchsin is used for elastic fibers; fast yellow TN is the component used for collagen and cytoplasm; pontacyl blue black SX is the nuclear stain. Procedure: Paraffin sections to water; aldehyde-fuchsin, 30 min; 70% ethanol; distilled water; 0.75% pontacyl blue black SX in 1.5% K.₂Cr₂O₇, 15 min; tap water; 70% ethanol to wash off all free dye; 2% fast yellow TN in 95% ethanol, 5 min; dehydrate, clear and cover.     

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.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Automated selection of the most epithelium-rich areas in gynecologic tumor sections.

The paper describes an image analysis technique for automated selection of the epithelium-rich areas in standard paraffin tissue sections of ovarian and endometrial premalignancies and malignancies. Two staining procedures were evaluated, Feulgen (pararosanilin) and CAM 5.2, demonstrating the presence of cytokeratin 8 and 18; both were counterstained with naphthol yellow. The technique is based on the corresponding image processing method of automated estimation of the percentage of epithelium in interactively selected microscope fields. With the technique, one image is recorded with a filter to demonstrate where epithelium and stroma lie. This filter is chosen according to the type of staining: it is yellow (lambda = 552 nm) for Feulgen and blue (lambda = 470 nm) for anticytokeratin CAM 5.2. When stroma cannot be distinguished from lumina with the green filter or from epithelium with the blue filter, a second image is recorded from the same microscope field, with a blue filter (lambda = 420 nm) for Feulgen and a yellow filter (lambda = 576 nm) for anticytokeratin CAM 5.2. Discrimination between epithelium and stroma is based on the image contrast range and the packing of nuclei in the yellow image and on the automated classification of the gray value histogram peaks in the blue image. For Feulgen stain the method was evaluated on 30 ovarian tumors of the common epithelial types (8 borderline tumors and 22 carcinomas with various degrees of differentiation) and 30 endometrial carcinomas of different grades.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.     

A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.     

A simple polychrome stain for conventionally fixed Epon-embedded tissues.

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO4 fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H2O2, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic fibers. Intracytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Visualization of DNA-containing structures in various species ofChlorophyta, Rhodophyta andCyanophyta using SYBR green I dye

We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine staining in vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxa Chlorophyta, Rhodophyta and the prokaryotic Cyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green alga Chlamydomonas reinhardtii and Scenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structures in vivo.     

Light microscopic triple-colored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique

The sequential application of the avidin-biotin-peroxidase complex technique was used to localize multiple tissue antigens on a single free floating section of rat brain. Sequential visualization of individual antigens was achieved by the silver-gold-intensified diaminobenzidine (DAB) in the first step, nickel-intensified DAB in the second step, and the DAB alone in the third step of the immunostain procedure. For the demonstration of this method, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF), and vasopressin (VAS) antisera were used. Sections from the hypothalamic paraventricular nucleus (PVN) of rats pretreated with colchicine were stained. Black TH containing cell bodies were clearly distinguished from blue stained CRF cells and from yellow stained VAS-containing cell bodies in the PVN on the 25-30 micron thick vibratome sections. The sequential immunostaining procedure presented here results in superior staining of multiple antigens as compared to that achieved by the sequential application of the peroxidase-antiperoxidase (PAP) technique.     

Chromatin Texture Analysis in Living Cells

In recent years, there has been an increasing interest in applications of fluorescence measurements to studies on many physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization. This type of approach involves both optimal conditions for DNA staining and the use of image cytometry. In this context, this report describes the application of an internal grey-level segmentation method for the assessment of real time modifications of chromatin organization in living cells. These developments are based on a specific, stoichiometric method for nuclear DNA content measurement. Preliminary data obtained from Hela cells suggests the possibility of following variations of nuclear texture (heterogeneity, granularity, condensation, radial distribution) related to the cell cycle progression of cells that are maintained alive.     

Uptake and accumulation of the vital dye hydroethidine in neoplastic cells

Hydroethidine, a reduced form of ethidium bromide, was used as a vital dye in fluorescence assays that allowed visual and semiquantitative monitoring of dye uptake and accumulation by fluorescence microscopy, flow cytometry, image analysis, and microfluorimetry. The excitation and emission filters were chosen to detect hydroethidine and exclude ethidium. Microscopically, there were differences in fluorescence intensities and fluorescence patterns among various tumor cell lines. The fluorescence pattern varied from homogeneous blue in the cytoplasm to blue plus brilliant packets of bluish-white distributed in the cytoplasm. Nuclear staining varied from brown to reddish orange fluorescence. These differences were confirmed by flow cytometry and image analysis. A preliminary survey of various tumors indicated that uptake and accumulation of hydroethidine were dependent on concentration of the dye, duration of cell exposure to the dye, and metabolic state of the cells. Microfluorimetry made possible monitoring of 96 samples in a microculture plate in 30 seconds; thus, this method allows large numbers of samples to be read, with a tremendous savings in time and reagents. The results obtained from the different techniques used were corroborative; therefore, any one of the above techniques may be used in an assay.     

Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei.

BACKGROUND: Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens. METHODS: Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data. RESULTS: The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample. CONCLUSIONS: The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.