Nuclear membrane, fixation

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... 

The stain/fixation method is usually used for surface markers that can withstand fixation and is followed by the application of a DNA-binding fluoro-chrome. The fixation/stain method is used not only for surface markers that can withstand fixation, but also for intracellular constituents, such as cytoplasmic proteins, nuclear membrane, and nuclear proteins. This is accomplished by using a crosslinking fixative (e.g., paraformaldehyde [PFA] or formalin) followed by a permeabilizing agent (e.g., Triton X-100, Tween-20, saponin, or lysolecithin). Some of the precipitating agents (e.g., ethanol, methanol, or acetone) can also be used for permeabilization after the initial fixation with PFA or formalin, or they can be used alone for both fixation and permeabilization (see Chapter 8). 

Sea Urchin. Immunofluorescence detection of lamins of sea urchin sperm nuclei and male pronuclei in vivo was originally reported by Schatten et al. (1985). Antibodies to lamin A/C and B were shown to label the poles of sea urchin sperm nuclei exclusively, suggesting that lamins were lost during spermatogenesis. Prior to fixation, however, these preparations were treated with 1% NP-40, a nonionic detergent that, tike 1% Triton X-100, has been subsequently shown to remove lateral nuclear membranes as well as lateral lamin components (Collas et al., 1995). 

Heat treatment, on the other hand, elevated cytoplasmic immunoreactivity of Bcl-2. However, nuclear and mitotic Bcl-2 immunoreactivity was clearly present when these cells were fixed with formaldehyde (3.6%), followed by postfixation with methanol for 10 min at -20°C. Treatment with ice-cold methanol makes the cell membrane permeable, allowing antibody access to intranuclear antigens without protein relocalization. Extensive protein crosslinking with formaldehyde is required for maintenance of intranuclear Bcl-2 immunoreactivity. In contrast to Bcl-2, Bax immunoreactivity was detected in nuclear and cytoplasmic compartments regardless of the duration of formaldehyde fixation used. 

In addition to preventing antigen elution or degradation, fixation also should preserve the position of the antigen, whether nuclear, cytoplasmic or membrane-bound, and preserve as much antigenic secondary and tertiary structure as possible, to provide a target for antibodies that will be used to detect the antigen. 

The JB67 mutant of Arabidopsis is characterised by an increased proportion of palmitic acid (16 0) and the unsaturated species of palmitic acid are correspondingly reduced in the thylakoid membrane lipids [3]. The defect has been shown to be the result of a single mutation of a nuclear gene. The reduced level of unsaturation of the 16-carbon acyl chains results in a slight decrease in CO2 fixation and reduction is the rate of photosynthetic electron transport but there in an increased thermal stability of the photosynthetic apparatus at elevated temperatures. 

There are at least two reasons that other embryo ftsation procedures might be used, however (1) Cytoskeletal elements, particularly microtubules, are not preserved well by the method above and (2) mutations affecting the chemistry of the vitelline membrane may preclude successful devitellinization. In either case, a one-step fixation/devitelliniza-tion in methanol/EGTA solution may be acceptable, although the nuclear morphology is not as well stabilized by this procedure. 

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