By G. L. Nicolson (auth.), James K. Koehler Ph. D. (eds.)
The use of the time period "advanced" within the name of this publication is a little ar bitrary and intensely a lot relative with admire to time. Many ideas which have been thought of on the "cutting facet" of ultrastructural technique quite a few years in the past are actually rou tin ely utilized in a number of laboratories. one can cite freeze-fracture, cryothin sectioning, or certainly lots of the box of experiment ning electron microscopy as concrete examples. therefore using the time period "ad vanced thoughts" has to be interpreted with reference to the current state-of-the-art, and comes in handy purely in informing the capability reader that this quantity isn't a primer for use as an preliminary creation into simple organic elec tron microscopy. many glorious volumes have stuffed that area of interest long ago few years, and it isn't meant that this modest e-book be a whole com pendium of the sector. in addition, any restricted number of papers on advanc ed thoughts unavoidably displays the personal tastes and arbitrary whims of the editor, thereby except many both very important systems which the a professional reader will without problems establish. the 1st quantity of this sequence seemed nearly 5 years in the past and illustrated ideas that have been concept to symbolize complex and but ba sically morphological equipment for gaining elevated ultrastructural informa tion from organic specimens. the current quantity, however, stresses concepts which offer particular physicochemical info at the speci mens as well as the structural information.
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Extra resources for Advanced Techniques in Biological Electron Microscopy II: Specific Ultrastructural Probes
1973; KOEHLER and PERKINS, 1974; KOEHLER, 1976; KOEHLER and KINSEY, 1977). , 1973). Although only a few examples for the ultrastructural use of labeled antibodies have been presented, it is clear that the use of labeled antibodies to attack biological problems can be a useful approach where there is a need to 40 W. D. PERKINS and]. K. KOEHLER identify a highly specific molecular species with a level of resolution beyond the limit of the light microscope. B. Antibody Labels For any research project that requires the use of labeled antibodies, it is important to carefully evaluate the label to be used since each has its advantages and disadvantages.
1972 b). However, if receptor site mapping of single cells is being considered, the two-step method may be more appropriate. General procedures for labeling with ferritin and HRP are dealt with in another Chapter of this volume (NICHOLSON, 1977). Therefore, it is the pur- 42 w. D. PERKINS and J. K. KOEHLER pose of this discussion to present methodologies for labeling antibodies with the markers hemocyanin and radioiodine for TEM examination. The labeling techniques to be considered are those most commonly used at the present time.
4) and washed three times (5 - 15 min each) in buffer. The fixed cells or tissue are reacted with Con A (1 mgl ml) at room temperature for 1 h and washed again three times for 5 to 15 min each 22° C. ) in phosphate-buffered saline is added and allowed to react with the Con A-labeled cells for 30 min at 22° C. Excess dextran-iron complex is removed by three washes as before, and the samples are postfixed in 1% osmium tetroxide in phosphate buffer for 1 h and dehydrated and embedded using standard procedures.
Advanced Techniques in Biological Electron Microscopy II: Specific Ultrastructural Probes by G. L. Nicolson (auth.), James K. Koehler Ph. D. (eds.)