In-situ hybridization . See

As the notochord is crucial for the development of the surrounding tissues but can be replaced by the floor plate of the neural tube (Fig. IB and IE), once this structure is established, the effect of notochord ablations varies depending on the type and timing of the operation. It is therefore advisable to analyze the embryos for the process of interest, but in addition for a known phenotype of the particular manipulation. This can be achieved, for instance, by double whole mount in-situ hybridization (see Fig. 1), employing molecular markers... 

Jiranek WA, Machado M, Jasty M, Jevsevar D, Wolfe HJ, Goldring SR, et al. Production of cytokines around loosened cemented acetabular components. Analysis with immunohistochem-ical techniques and in situ hybridization, [see comment]. J Bone Joint Surg Am 1993 June 75(6) 863-79. 

This protocol has been through numerous generations of modifications by many laboratories, but it is based primarily on Tautz and Pfeifle (1989). All incubations and washes can be carried out in 1.5-ml microcentrihige tubes. For X-gal staining followed by in situ hybridization, see Protocol 12.5 Notes. 

Postmortem human brain tissue is obviously not subject to as rigorous control as experimental tissue from laboratory animals. As such much care needs to be taken when attempting in situ hybridization with human brain. In addition to the normal factors such as age and gender that need to be controlled with human samples, other factors, such as the immediate premortem state of the individual, the time taken for tissue recovery, and the storage conditions, must also be considered (for review, see Hynd et al., 2003). 

Early investigations of virtually aU nemonal nAChR subunit genes, included in situ hybridization studies that determined mRNA expression patterns in rat [see, for... 

If cellular localization of the antigen-antibody complex is not required, enzyme immunolabeling can be performed on cells adherent to a microtiter plate, and the color change resulting from the enzymatic reaction can be detected as a change in absorbance with an automatic plate reader (see Chapter 28). Biotinylation of antibodies and the use of the avidin-biotin complex has further extended the versatility and sensitivity of the enzymatic techniques (see Chapters 7 and 25-27). Most recently, the principles behind these techniques have been applied in combination with in situ hybridization techniques. Using nucleic acid-antibody complexes as probes, specific DNA or RNA sequences can be localized (see Chapters 46 9). 

Proper fixation is one of the most critical steps in an in situ RT-PCR or in situ hybridization experiment, because each tissue type must have optimized fixation conditions. Particularly archival tissues may require individual specific treatment in order to meet with in situ experimental requirements. Errors in fixation will only be discovered after the entire hybridization process has been completed (see Note 10). 

Fig. 4. In situ hybridization technique. Part of an erythroid precursor cell infected with the human parvovirus B19 and probed for viral DNA. The BI9 nucleic acid is located within the centra electron lucent area of the nucleus (N) and also at nuclear pores (arrowheads). Ch, chromatin M, mitochondrion. A three-step detection protocol was used (see ref. 5 for details). Sheep antidigoxigenin followed by rabbit antisheep Ig and then goat antirabbit Ig conjugated to 10-nm gold. Bar is 0.5 pm.

Fig. 5. Combined in.vi hybridization and immunocytochcmistry. Part of the nucleus of an erythroid precursor ceil infected with parvovirus B19. There is colabeling of the viral DNA 10-nm gold) using in situ hybridization and the B19 capside protein (5-nm gold) by immunocytochemistty over an intranuclear crystalline array (Cr) of viral particles see ref. 7 for details). Nu, nucleus Ch, heterochromatin. Bar is 0.1 pm.

In situ hybridization overcomes the problem of cellular localization, but it is difficult to relate the expression of a particular mRNA to the expression of the functional protein. Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand, is a relatively simple technique that overcomes these problems by identifying the precise cellular localization of the functional protein. This technique, using paraffin sections, provides information on the ER status of tumors very simply and rapidly. In addition, this approach is superior to frozen section immunohistochemistry, the dextran-coated charcoal assay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) for predicting the response to endocrine therapy. 

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