Cell fixation tissues

There are a number of different cell fixation and process methods used in laboratories worldwide. Fixation time in tissue will reflect a commonly accepted fixation time such as that seen in pre-analytical guidelines published in the package inserts for commercially available kits. Regarding cell lines points to note are how soon are the cells fixed after harvesting, are the cells fixed in suspension, or when are they in a suspension matrix such as agarose. 

Finally, the localizations of low-molecular-weight compounds requires special specimen preparation techniques, as these compounds are often diffusible, water- or organic-solvent soluble, and solubilized by conventional fixation and dehydration procedures. The reader is referred to ref. (12) for the processing of cells and tissues for the cytochemical and histochemical localization of these compounds. 

List of Fixation Conditions for Preparation of Plant Cells and Tissues for Transmission Electron Microscopy... 

As mentioned, chemical fixation of plant cells has been reviewed many times (15-20) and the reader is referred to these citations for a variety of fixation procedures for preserving plant cells and tissues. One of the most recent references regarding the topic is that of Hopwood and Milne (21). Table 1 presents their recommendations regarding fixation of plant cells and tissues for electron microscopy. 

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... 

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immuno-localization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immuno-localization at the level of light microscopy. 

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. 

Cellular and nuclear localization of autoantibodies was initially described three decades ago (A7, A8, A9). However, some argued that they only represent a fixation artifact, with movement of immunoglobulins into cells during tissue preparation... 

Comparisons have been made of HA localization in skin sections fixed with acid-formalin/ethanol and conventional formalin fixation. Much of the HA, particularly in the epidermis, is eluted during the process of formalin fixation. This suggests that epidermal HA is more loosely associated with cell and tissue structures than is dermal HA. A further incubation of 24 h in aqueous buffer further increases the disparity between the acid-formalin/alcohol and the conventional fixation technique. Once the tissue has been exposed to the acid-formalin/alcohol, the HA association with tissue becomes tenaciously fixed, with little loss of apparent HA observed following additional aqueous incubation, while the formalin-fixed tissues demonstrate progressive loss of HA. 

Biological autofluorescence in mammalian cells due to flavin coenzymes (FAD and FMN absorption, 450 nm emission, 515 nm) and reduced pyridine nucleotides (NADH absorption, 340 nm emission, 460 nm) can be problematic in the detection of fluorescence probes in tissues and cells. Fixation with aldehydes, particularly glutaraldehyde, can result in high levels of autofluorescence. This can be minimized in fixed cells by washing with 0.1% sodium borohydride in phosphate-buffered saline (5) prior to antibody incubation. Problems due to autofluorescence can be minimized by selecting probes and optical filters that maximize the fluorescence signal relative to the autofluorescence. Other factors that limit IF include the performance of the detection instrument (i.e. how well the microscope has been calibrated and set), the specificity of the antibodies, and the specimen preparation. 

The cells or tissue sections are then fixed to preserve cellular structure and immobilize proteins in place. Fixation is often accomplished by incubating the cells/tissue in organic solvent, paraformaldehyde, or glutaraldehyde. 

Since protein complex formation and Ca2+ are critical to cell fixation within a tissue, dissociation media usually contain some type of proteolytic enzyme and the Ca2+ chelator, EDTA. The proteolytic enzyme can be of general specificity, such as trypsin, or can be a more targeted enzyme, such as a collagenase selective for the collagen-type characteristic of the tissue of interest. Hyaluronidase has been also used with matrix rich in hyaluronic acid, such as for isolation of duodenal entero-cytes. In all cases, the appropriate incubation times and concentrations to achieve cell dispersal, but retain high viability, need to be determined empirically. One factor... 

To allow efficient extraction of intact mRNA, it is recommended that alcohol fixatives be used in lieu of cross-linking agents such as paraformaldehyde. In our experience protocols that have been developed for studying paraformaldehyde-fixed paraffin-embedded archival specimens in tandem with RNA amplifications produce low yields of RNA from microdissected brain regions and picked cells. Fixation of tissue with methanol or ethanol preserves immu-noreactivity and mRNA in many cases. Accordingly, we immerse... 

Fixation of an implant in the human body is a dynamic process that remodels the interface zone between the implant and living tissue at all dimensional levels from the molecular up to the cell and tissue morphology level and at any time scale from the first second up to several years after implantation (Kasemo and... 

Sample preparation is one of the most important steps in immunocytochemistry because it generally receives the least amount of planning. Unfortunately, few researchers understand how critical the first few steps in an immunocytochemistry experiment are. In fact, the quality of the cells and tissue and the ability to get good results are totally dependent on initial fixation. To put sample preparation in perspective, perhaps the best thing to do is focus on the conclusion of the experiment -the quality of final microscopic image. Good images only come from cells and tissues that are fixed properly. 

Fixation is the stabilization or preservation of cells and tissue as close to life-like as possible. All fixation procedures change the tissue they are preserving, but the key is to find the least amount of change for immunocytochemistry. 

Ideally for immunocytochemistry, it would be nice to examine live cells and tissues without any fixation. The problem with looking at cells and tissue in the microscope is that the sample must be thin enough to examine in the microscope and it must be stable enough so as not to deteriorate while being examined. These criteria require the use of fixatives to preserve the cells and tissues. 

In theory, fixation for microscopy is based on the need to obtain images of tissues and cells as they were when living, with no changes or distortions. However, fixation has the potential of introducing changes in cells and tissues. The purpose of this chapter is to guide sample preparation to minimize unwanted changes to cells and tissues. 

The following are the criteria for good fixation. Keep these in mind when evaluating cells and tissue sections. 

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