Double embedding procedure

Although several techniques are available for the determination of refractive index, only the double embedding procedure has been used with wood specimens (Boutelje 1972, Donaldson 1985a). This procedure involves measuring optical path difference (o.p.d.) successively in two different reference media. Selection of reference media depends on a number of criteria ... 

For the double embedding procedure, refractive index can be calculated from o.p.d. values using the following equation ... 

The IEM double-labeling method described was performed with cells fixed in 2% paraformaldehyde and 0 04% glutaraldehyde, and embedded in Lowicryl K4M, LR White, or LR Gold. Other fixation and embedding procedures should work equally well, provided that the specimen resists the chemicals used m the silver enhancement. 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

The avidin-biotin technique is especially useful in double-labeling experiments. This can be done using two different enzymes in the final step, combined with two different andbody species (monoclonal and/ or polyclonal). One of the andbodies can be biodnylated and the other immunochemical reaction can be based on a different procedure (e.g., employing an antibody-enzyme conjugate). Alternatively, both antibodies can be biotinylated and the secondary reaction with the respective avidin-enzyme conjugate can be performed on different sides of an impermeant (e.g., plastic-embedded) tissue section. 

While the embedding orbitals are kept frozen, we supply on the atoms to be correlated an extended basis set of at least double- quality with polarization functions, recalculate the integrals and perform an HF calculation for the orbitals to be correlated. During this HF calculation the central orbitals are reoptimized and can develop a metallic character. But the long-range tails are absent, because the embedding region is kept frozen and no / -functions are supplied. This procedure is quite critical for metals and has to be tested carefully. Sec. 3B will deal with this problem in detail. 

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