Special Applications of In Situ Amplification

3.5.1. In Situ Amplification and Immunohistochemistry

1. Fix cells or frozen sections of tissue, which are already placed on slides, with 100% methanol for 10 min.

2. Wash slides in PBS.

3. After that, labeling of surface antigen(s) can be carried out by standard immuno-histochemical method: FITC-conjugated antibody is incubated for 1 h at 37°C, washed, and then cells or tissue section are fixed in 4% paraformaldehyde for 2 h.

4. In various pathology laboratories, many specific surface antigens have been tabulated that can withstand 10% formalin and other routine histopathology procedures and will still bind to specific monoclonal antibodies. If one is using any of these immunohistochemistry panels, then one can also use routinely prepared paraffin sections for the detection of cellular antigens.

5. Then, the tissue is prepared for in situ amplification, as described previously.

3.5.2. Multiple Signals, Multiple Labels in Individual Cells

1. As described in the previous section, one can label proteins by FITC-labeled antibodies.

2. Then, one can perform both RNA and DNA in situ amplification in the cells.

3. If one is using primers for spliced mRNA and if these primers are not going to bind any sequences in DNA, then both DNA and RT amplification can be carried out simultaneously.

4. Subsequently, products can be identified by using different color probes, with different excitation and emission ranges, resulting in different colors of signal. Currently, there are more than 30 different kinds of color probes available, and one can choose them according to the filter range one has in the laboratory.

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