Fixation Procedure

After harvesting by the appropriate method, ceU suspensions can be fixed by a number of different procedures depending on the application (Protocol 1). The 

Add 3 ml of absolute methanol, cooled to —20 C, dropwise whilst vortexing. 

Dilute to 70% with distilled water and store at 4 C until required. 

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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. 

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

VieUdnd, U. and S wierenga, S. H. (1989) A simple fixation procedure for immuno-fluorescent detection of different cytoskeletal components within the same cell. Histochem. 91, 81-88. 

Despite the harshness of these reagents (see Note 1), the organic solvents are the simplest and most rapid method to fix and permeabilize cells. Methanol is preferable to ethanol in many cases. The basic fixation procedure outlined below is suitable for the detection of total PCNA within the cell and for Ki-67 in most cells. 

Practically speaking, that would mean that overnight fixation would be required, that weekend fixation would be OK, but a longer fixation time would not be useful. With this information, users would be able to evaluate the results obtained from tissues received from outside sources by comparing fixation procedures. 

This book will focus on the chemical finishing of textiles, the application of relatively minor amounts of chemicals (often < 5 g m" ) to, in most cases, both sides of the fabric. Subsequent chapters will discuss the importance of each specific finish, the chemical mechanism for the effect, the chemicals used to provide the desired properties, the apphcation and fixation procedures, the relevant evaluation methods and trouble shooting tips. Processes that employ high levels of chemical apphcation (15-50 g m and more), primarily as one-sided treatments, such as coating are addressed only briefly in Chapter 2. 

As can be seen from the data in Fig. 7, carboxylesterase dissociates at a slow rate even at pH 7.4. In the presence of salt, this dissociation is increased. Dissociation in the presence of salt was also measured by the complement fixation procedure. In Fig. 8 are shown the equilibrium constants for dissociation in varying concentrations of KBr and LiBr at pH 7.2 (10 mM Tris). 

Figure 7.27 A cubic membraiie that seems to uitfold asymmetrically. Note such effects can in principal be introduced as an artefact due to fixation procedures, and the (asymmetric) osmotic pressure present under these circumstances. From [104], with permission.

A marked degree of variability in the immunohistochemical results has been demonstrated in different breast cancer studies (56-58). The degree of positivity varied from 15.5-54% in 14 breast cancer studies (57). The number of immunohistochemically positive cells varied from 29-54% with four different p53 antibodies (59). Dissimilar fixation procedures and different paraffin temperature may provide explanation for the partly different immunohistochemical results (60). The importance of these issues have been underlined in a pilot study on 22 breast cancer biopsies using the mAh pAb 1801 for p53 determination. Formalin was compared with Bouins fixative, with no difference in the results obtained (37,60). However, the fixation time was of importance. Six h formalin fixation was compared with 24 h fixation. Fifteen of 22 samples fixed for 24 h completely lost their immunoreactivity. Interestingly, microwave treatment retrieved the p53 antigen in all cases but one (60). 

SpA-containing Staphylococci (Section 3.3), fixed with trichloroacetic acid (TCA) or formalin (Section 3.3.1), may also serve as immunosorbent for many mammalian antibodies (Table 7.1). Both fixation procedures are satisfactory but yield products with different properties. Fixation of Staphylococci with hot TCA (Lindmark, 1982) removes the negatively charged cell-wall polymer teichoic acid, producing an IgG-sorbent which can bind 1.4 mg human IgG per ml of a 10% (v/v) suspension of bacteria and is stable for about 5 months. Formalin-fixed bacteria (Kessler, 1976) bind 35% more IgG and are stable for at least 1 year. However, IgG can be eluted quantitatively from TCA-fixed bacteria but not from formalin-fixed bacteria, probably due to the interaction between IgG and teichoic acid, unless 80 mM MgCh is included in the acid buffer. 

Carefully, remove the PBS from the microcentrifuge tube and start the fixation procedure (see Immunocytochemistry of Imaginal Discs and Pupal Retinas ). 

Due to the penetration properties of a few antibodies, in some cases it is better to also remove the optic lobes before starting the fixation procedure. 

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. 

The five fixation procedures were evaluated systematically to compare preservation of adduct antigenicity in immunostained sections and cell structure in hematoxylin and eosin (H E)-staiiied sections (Table I). 

It should be noted, fast of all, that in all these fixation procedures, we can substitute, for bacterial bodies the extracts of these substances or toxins, as antigen. Thus tuberculin permits the examination of the antibodies contained in the serum of tuberculous subjects. Likewise for syphilis, the extracts of syphilitic organs can be used as antigens, and in the presence of the antibodies contained in the serum of infected individuals, will produce the fixation. Since the publication of the work of Wassermann, there have been numerous modifications introduced... 

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