Cell cultures immunocytochemistry

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. 

Fig. 4. Immunocytochemistry of formalin fixed 147L) cell cultures using a-PR-A anti-receptor MAb (left) and a control MAb (right). Immunocytochemistry was performed with T47D breast cancer cells grown as monolayers in chamber slides. Cells were fixed for 15 min with 3.7% buffered formalin, followed by a permeabilization step with Triton X-100 (0.1%) for antibody penetration into the cell. Immunocytochemistry was performed by the indirect avidin-biotin immunoperoxidase method using diaminobenzi-dine as the chromagen.

In research, a technique which has evolved in importance is the analysis of the living cell as seen in cell culture. It is within the special boundaries of cell culture that conditions can be manipulated in order to examine living cells and to better understand their behavior (1). The fact that transformed or malignant cells grow very nicely in rather simple conditions of chemical formulation make these cells ideal laboratories to study cellular processes. Immunocytochemistry as applied to cell culture has allowed... 

Today, for research with animal tissue and cell cultures, the standard has become fixation in paraformaldehyde, with animal tissue sectioned in a cryostat, and then incubation of sections and cultures with antibodies. This book focuses on introducing the methods of immunocytochemistry for biomedical scientists. These chapters may be read in order for a complete understanding of immunocytochemistry, or the chapters may be read individually for information about specific topics. The book is designed to help the novice perform experiments, solve problems, get results, and understand more advanced texts when more advice is needed. 

Fig. 4.7 Chamber slides for cell cultures. To grow cultured cells on a tissue culture plastic substrate for microscopy, one choice is chamber slides. The individual wells can be used to plate cells and process cells for immunocytochemistry.

Fc receptors in tissue bind the Fc end of the primary antibody and lead to incorrect labeling (Fig. 5.2). Fc receptors are found on macrophages and other immune cells, and can bind any antibody in the immunocytochemistry procedure. The primary antibody would most likely bind open Fc receptors as the first antibody incubation. This problem occurs only in areas where there are macrophages, such as injury and inflammation sites. Unless cell cultures include macrophage or natural killer cell lines, Fc receptor binding does not occur in cultures. 

Organic solvents like methanol or acetone also can be used to open membranes, but they also act as denaturing fixatives. Solvents are used at -20 C for 10 min, but the use is not recommended. As described in previous sections, denaturing fixatives may cause loss of epitopes in the cells. Other detergent-like agents, such as saponin or digitonin, are used for transient permeabilization of cell cultures, and reversibly insert into and out of plasma membranes next to cholesterol. These agents must be present in all solutions after the fixative and should only be used for electron microscopy immunocytochemistry. 

IDENTIFICATION OF CULTURED CELLS BY IMMUNOCYTOCHEMISTRY (INDIRECT IMMUNO-STAINING)... 

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... 

Fig. 3 Maturation of mixed neuronal-glial cultures of cerebellum granule cells (CGCs). Cell morphology (a and b) in phase-contrast microscopy and by immunocytochemistry of GFAP (red) and NF-200 (green) co-stained with Hoechst 33342 (c and d)...

If the terms immunocytochemistry and immunohistochemistry seem similar then here is why. Many years ago, immunocytochemistry was defined as the use of antibodies to study cells in the form of cultures or smears from animals. Immunohistochemistry, on the other hand, was defined as the use of antibodies to study paraffin sections from human tissue. Today, immunohistochemistry is still... 

Another example of a population study that uses antibodies is flow cytometry. Isolated cells must be dissociated from tissues or cultures and labeled with fluorescent antibodies specific for a subpopulation of the cells. In flow cytometry, cells pass rapidly past a detector that measures the amount of fluorescence for each cell. The size of cells and the amount of fluorescence can be plotted and analyzed. Even though this method makes use of antibodies, it is a population study because it determines the number of isolated cells bound to an antibody. Flow cytometry identifies different populations of isolated cells, but it cannot show the location of these labeled cell in tissues, which can be done only with immunocytochemistry. 

Here then is how it all works together. Immunocytochemistry takes tissue sections or culture cells and incubates them with antibodies. The experimental needs determine the exact order of antibodies incubations and the specific labeling of the antibodies. The general steps in a single primary (1°) antibody indirect immunocytochemistry experiment include the following ... 

This book is intended for scientists who are working on research animals and cultured cells. The procedures described here give the best results with the easiest methods. Note that many older procedures and reagents are still used today, but they give less than ideal results. For example, the fluorophore FITC was the first fluorophore used for immunocytochemistry by Albert Coons in 1942, when he invented this field. Since then, three new generations of fluorescent compounds (Chapter 6, Labels) with improved photobleaching properties have evolved making FITC of historical interest for immunocytochemistry. 

Antibodies can come in a variety of forms and purities. Polyclonal antibodies can come as whole serum or as purified antibodies with an IgG concentration of 1 mg/ml. Monoclonal antibodies come as isolated tissue culture media from hybridoma cells called supernatant. The antibody from supernatants is between 50 and 100 tig/ml, which means that the working antibody dilution for immunocytochemistry will be lower than whole serum. In addition, monoclonal antibodies can be ascites fluid giving antibodies that are highly concentrated of 1 mg/ml. Today, generation of ascites may be restricted by federal regulations for care of research animals. 

All tissue and cultures for immunocytochemistry must be fixed to preserve them. Unfixed cells and tissue degenerate quickly, leaving nothing to be seen. In fact, once the tissue culture medium or blood supply is removed, the process of degeneration begins. Therefore, it is crucial that immediately upon removing the tissue from the animal or the cells from the culture medium begin the preservation process with a fixative. 

Chamber slides are best for cells that require tissue culture grade plastic as a substrate. Lab-Tek chamber slides come with such a slide and they come in a range of well sizes, but a very practical size is the 4-well plate (Fig. 4.7). The microscope slide base is made of Permanox, the same plastic used for plastic petri dish bottoms so that the cells grow like they do on petri dishes or flasks. The nice thing about the chamber slides is that wells on the slide do not need be removed until after immunocytochemistry processing. The wells can be removed just before mounting a... 

A new Experimental Design Chart must be used for this Indirect Immunocytochemistry Block-Between (Table 12.1), because it differs from the chart used previously. Here, the experiment uses cultured cells on coverslips and two 1° antibodies made in mouse, mouse anti-Ag A and mouse anti-Ag B. A new row on the Chart is needed, 4. Block-between, to list two new reagents used to block-between the two 1° antibodies. Normal mouse serum is used at 1 20, which is sufficient to block in most cases. The anti-mouse Fab molecule is used at 20 p.g/ml. Incubations for each of these steps is for 1 h with six rinses after each incubation. Note these blocking incubations must be performed in the order of normal mouse serum first and anti-mouse Fab second. 

Postembedding electron microscopic immunocytochemistry uses tissue or cultured cells that are chemically fixed, sectioned on a diamond knife, and processed for immunocytochemistry. Here, all procedures and reagents, prior to... 

Transfection techniques in vitro and ex vivo (organotypic cultures) offer an array of possibilities to investigate the consequence) s) of the introduction of foreign nucleic acids (DNA but also RNA) and or other biologically active molecules into neurons, and to combine observations with immunocytochemistry. In particular, a wide number of fluorescent reporter proteins (FRPs) can be employed for multicolor fluorescence imaging. Here we present a series of protocols for in vitro and ex vivo transfection of DNA or RNA sequences into cerebellar neuron cultures and organotypic slices based on the use of plasmid vectors and multicolor laser scanning confocal microscopy. These protocols allow analysis of live transfected cells, and, after fixation, correlative neurochemical studies. 

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