Cell fixation technique

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. 

In-Cell Western (Li-COR Cytoblot) Anti-phospho specific antibody labeled with infrared fluor is used to probe fixed cells on MTP second color can be used for normalization binding detected via laser scanning technique Only requires one antibody has potential for multiplexing to control for total substrate expression in cells Multiple wash steps requires cell fixation lower throughput Chen (2005)... 

Use hypotonic treatment/fixation technique, and affix cells to slide Treat cells with RNase (and/or proteinase K)... 

Some patients with lymphocytic thyroiditis have antibodies directed against the microsomal or cellular fractions only. Others have positive results for the thyroglobulin antibody, but have no antibodies against the microsomal fraction. The microsomal antibody is the most significant in relation to the etiology of thyroiditis as it is cytotoxic to cultured thyroid cells (D6). Microsomal autoantibodies are detected either by complement fixation technique (CFT) or by immunofluorescence. Be-... 

Artifacts may be caused by the migration of proteins and low molecular compounds within the cell because of increased membrane permeability with cell death. In most cases, therefore, it is necessary to use appropriate fixation techniques. Enzymes, for instance, can be bound to particulate cell constituents by glutaraldehyde. The fixation of low molecular weight substances, however, is still difficult. In the case of hydrophilic compounds, diffusion after cell death may be prevented either by rapid freezing and subsequent exchange of the cell... 

Mucous cells, found in the neck of the gland, secrete a mucus with staining properties different from those of the surface epithelium. Argentaffin cells are not seen with ordinary staining or fixation techniques. After fixation with potassium bichromate and silver or chromium impregnation, they stand out as cells scattered singly between the lining of chief cells and the basal membrane. 

Additional double-stranded RNAs were discovered to be interferon inducers. The activity of a DNA-RNA hybrid synthesized from single-stranded DNA of f phage was demonstrated by complement fixation techniques to be entirely due to contaminanting double-stranded RNA. Other active double-stranded RNAs were Isolated from normal mammaHan cells. 

Protocol 5.2 describes a methanol-acetone fixation technique (Pisano et al. 1993 Cenci et al. 1994) which results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics exhibit most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation protocol results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is a poor preservation of chromosome structure. In most instances, the chromosomes do not exhibit a distinct morphology and tend to coalesce into one or more masses of chromatin. 

Immobilized cell Technique used for the fixation of enzymes or cells onto a solid support. 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

In essence, the basic steps of making cell blocks consist of fixation, centrifugation to make cell pellet, transfer the pellet to a labeled tissue cassette which then is processed and embedded in paraffin. The most challenging component of this technique is the methods to harden the cell pellet so it can be easily picked up from the tube without losing precious material. With only a simple sedimentation technique, the cell pellet is usually small and friable. In order to harden the cell pellet, several technical modifications have been reported. The most popular methodology includes plasma-thrombin clot technique, agar technique, and fixation with Bouin s solution. 

Standardization of IHC/ICC has been a critical issue for more than three decades, especially with the advances in targeted therapy such as the development of trastuzumab (Herceptin) for advanced breast cancer.51 Nevertheless, standardization is a difficult issue because numerous factors may influence the consistency and reliability of immunostaining results, including fixatives, fixation time, AR, antibody clones, detection system, and interpretation (see Part II). In cytopathology, the situation is even worse due to its variable cell sample preparation techniques. Cytopreparation is. .. the foundation of cytomorphology. 52 We believe it is also the foundation of ICC. Therefore, standardization of ICC needs to start with uniform and reliable cytopreparation. 

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. 

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. 

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