Sample preparation/fixation procedure

Depending on the concentration of DNA used in the sample preparation procedure, the surface coverage can be adjusted. The fixation using the silanization procedure is beneficial in effectively immobilizing the DAN for AFM work. Stability in excess of 5 h has been reported [106] (Fig. 3.50). 

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

The basic procedure of biological sample preparation for SEM involves in specimen fixation, dehydration, drying, and coating (see Ref 27 for a comprehensive review of procedures). The purpose of fixation is to immobilize the cellular components, such... 

We therefore present two different procedures for fixing and preparing yeast samples for FISH. The first of these is suitable for rDNA hybridization and/or tubulin immunofluorescence (preservation of tubulin requires high concentrations of formaldehyde and rapid fixation). The second protocol is designed for hybridization with sequences of lower copy number and r immunofluorescence with antibodies to nuclear proteins such as RAPl or nuclear pore components. These antigens are extremely sensitive to overfixation, and so the cells must be fixed lightly for immunostaining, then postfixed for hybridization. If possible, use the first procedure because it is simpler and more reliable. 

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