Fixation tissue cultures

A complete assay requires the test material to be investigated at a minimum of three doses together with a positive (untreated) and solvent-only control can be omitted if tissue culture medium is used as a solvent. When two fixation times are used in repeat tests, the positive control is necessary at only one time but the negative or solvent control is necessary at both times. 

Fix the specimen. For this example, the live coelenterate Hydra oligactis is first relaxed using an anesthetic solution of 2% urethane in hydra culture medium for 1 min to prevent contraction into a ball on fixation (17). 0.5 mL is used in a well of a 24-well tissue-culture plate. 

The most common and simplest procedure is to place a few microliters of the test solution over a small puncture wound on a detached leaf. The puncture wound enhances the access of the toxin to the leaf tissue. The leaf is then placed in a petri dish containing a filter paper saturated with water. The top cover of the plate is sealed with parafilm, and the plate is incubated under controlled light and temperature conditions. Toxin activity is usually indicated by chlorotic, necrotic, or colored spots on the leaf. Other methods for bioassay involving CO2 fixation, or effects on organelles, whole plants, protoplasts, tissue cultures, or plant parts are outlined (, 7). 

After treatment, the exposure mixture was removed and cells were grown for 18 h at 37 °C in medium plus serum. Then, 1 /zg of colcemid (Gibco) was added per tissue culture flask, and cells were incubated for another 2 h at 37 °C. The collection of the cells by trypsinization, their fixation, and the staining of chromosomes with Ciemsa were carried out according to standard procedures (18). At least 50 metaphases were screened for chromosomal aberrations per treatment group. 

Frozen material or tissue-culture cells may be fixed by immersion for 10 min in 50% methanol/50% acetone at -20°C, followed by air drying. Alternatively, a 4% solution of paraformaldehyde can be prepared by dissolving 4 g of paraformaldehyde powder in 80 mL of water with gentle heating and the addition of 1 M NaOH until the powder dissolves. The solution is made up to 90 mL with distilled water, and 10 mL of a 10X PBS solution added. Fixation is for 10 min at 4°C, and slides are then rinsed in PBS. Tissues or cells which are to be used for TUNEL/ISEL should be fixed as quickly as possible, because delay causes significant artifacts. 

The first step for a successful in situ hybridization is the fixation of the tissue. This will ensure target nucleic acid retention and preservation of the tissue morphology. Either crosslinking or precipitative fixatives can be used, and a preference for either of the two types of fixative has often been based on the different types of system under investigation (1-7). For hybridization of regulatory peptide mRNA, 4% paraformaldehyde appears to be the most effective, both on tissue blocks and on tissue culture preparations. 

The lesions seen in humans are indistinguishable for both orf virus and milker s nodule. They begin as a small red or reddish blue papule that enlarges to form a flat-topped hemorrhagic pustule, often crusted in its umbilicated center. Diagnosis is made by clinical observation and by means of the complement-fixation test, tissue culture, or electron microscopy. 

McLaren, ., Thomas,D.R. CO2 fixation, organic acids and some enzymes in green and colourless tissue cultures of Kalanchoe crenata. New Phytol. 66,683-695 (1967) McWilliams, E.L. Comparative rates of dark CO2 uptake and acidification in Bromeliaceae, Orchidaceae, and Euphorbiaceae. Bot. Gaz. 131,285-290 (1970)... 

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Blood smears, tissue imprints, cell cultures and purified cells may be examined as fresh tissue or as fixed tissue. These cells can be centrifuged to make a pellet that is then fixed just as in tissue fixation. Alternatively, a fresh smear may be made on the slide, and the cells fixed either with acetone or 10% NBF for 10 minutes. It is important to incubate the slide with an endogenous peroxidase blocking solution... 

After fixation (see above), cultured cells are incubated in stain solution containing X-gal (1 mg/ml, final) overnight at 37°C. Animal tissues are incubated in stain solution with X-gal (1 mg/ml, final), 0.02% (vol/vol) nonidet P-40, and 0.01% (wt/vol) sodium deoxycholate overnight at room temperature. Following staining, cultured cells are rinsed three times with PBS, while animal tissues are rinsed... 

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