Slide preparation fixed cells

In this test, cultured cells are seeded onto slides and the cells, which had been treated with and without metabolic activation for a short time period (e.g. 3 hours). Where negative or equivocal results are obtained, an independent experiment is conducted in which cells are treated for a long time period (e.g. 20 hours) in the absence of metabolic activation alone, and then sampled and examined for chromosome analysis. In both experiments the cells were sampled 20 hours after the start of treatment, as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 hours before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined. However, if clearly positive results were obtained in the first experiment, those from the second assay were not examined. If equivocal or negative results were... 

FITC-dextran FITC complexed to dextran used for observation and quantification, e.g. plasma extravasation, fixation A method using a fixative to kill cells but to preserve their structure and organization. A stage in microscope slide preparation. 

Many assays of apoptosis by LSC are performed on fixed cells. For these assays the cells are attached to microscope slides by standard methods that include smear films, tissue sections, touch preparations from freshly transected tissues, or cytocentrifuging cell suspensions. Cytocentrifugation (see below) is often preferred over touch or smear preparations because it flattens cells on the slides so that their geometry is favorable, and therefore more morphological details can be revealed. 

In preparing cells for cytospin slides, there are times when immediate fixation is necessary. For these instances, the cells can be fixed before centrifugation. Starting at step 2 for the cytocentrifuged specimen (Subheading 3.3.), continue with ... 

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.)... 

Liver cells are then exposed in vitro to the test compound and incubated with tritium-labeled thymidine for about 18 hours. At the end of the incubation, the cells are fixed on slides and prepared for autoradiography. For that the slides are first exposed to liquid photographic emulsion, air-dried and following a 7-day exposure in the dark, exposed to developing solution. 

Histopathological analysis was performed according to a published procedure by Lipkin. The entire cecum, colon, and rectum of two animals, one each from the control group and the MCM-treated group, were removed and fixed with 10% buffered formalin (12 h), 80% ethanol (12 h), and 95% ethanol (12 h). Representative sections were taken, paraffin embedded, and 4-pm sections cut, mounted into glass slides, and stained with hematoxylin and eosin. Five slides were prepared from each tissue, each slide containing five serial sections. The number of epithelial cells per intestinal crypt over 50 intestinal crypts was counted. The number of crypts containing dysplastic epithelial cells per 50 intestinal crypts was counted. 

The purpose for the preparation of these types of slides is to examine the cells of a tissue quickly, with no need for tissue preparation and cutting. It is important to work quickly since the tissue will start to deteriorate the moment it is obtained. Therefore, it is important to have all the preparations ready for the rapid handling of the excised tissue. As soon as a surface of the tissue is decided on, it should immediately be touched to a waiting slide, oriented properly, and fixed. As always, when handling fresh tissue, it IS imperative to wear gloves and protective clothing. 

The reference cell thus prepared is then set on the pedestal which is stuck at the center of the upper plate of the laboratory jack, which in turn is fixed on the laboratory desk. The position of the stand, by means of which the stem including the glass vent tube is held in the vertical position, is next adjusted by sliding the stand horizontally on the desk in order that the glass vent tube may be aligned vertically with the reference cell set on the pedestal. 

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. 

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