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Answers for December 01, 1997 Questions:

Figure #1 is background and Figure #2 is target specific signal.

The way to make this distinction is to have some knowledge of histology. Note that the staining pattern in Fig. #1 is around empty spaces, not in the cytoplasm of a cell (the spaces are areas between collagen bundles). This background was, for demonstration purposes, induced by omitting the essential post RT PCR high stringency wash (60C for 10 minutes with 0.2XSSX and 2% BSA). The labeled primer dimers that formed in the overlying solution nonspecifically stuck to cellular proteins and gave this background.
Note in Fig. #2 that the signal, after a high stringent wash, is seen in the cytoplasm of the tubular cells, as would be expected in human mRNA. The background is no surprise to anyone who does in situ or immunohistochemistry, where washing is essential to remove nonspecifically bound probe/antibody to cellular proteins and/or nucleic acids. Some people incorrectly refer to this background as back diffusion. Under optimal conditions of protease digestion and stringent washes, it is easy to show that the signal localizes only to the target cell by, for example, using as a model a virus which only can attack specific cell types. Parvovirus is such an example, as it infects nucleated red blood cells.

Figures 3 shows the H&E of a liver with giant cell hepatitis in a child who died of parvovirus infection; note the extramedullary hematopoesis where nucleated red blood cells are evident. After RT in situ PCR for parvovirus using optimal protease and a high stringent wash, these target cells are positive and the adjoining liver cells, which the virus does not attack directly are negative. (See Figure #4).

Figure #3

Figure #4

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