COMPARISON OF HISTOLOGIC STAINS FOR USE IN PCR ANALYSIS OF MICRODISSECTED, PARAFFIN-EMBEDDED TISSUES

We evaluated the effect of six different histologic stains on the prod uctivity of PCR amplification of DNA isolated from paraffin-embedded t issue samples. The tissue was collected from glass slides by microdiss ection techniques, whereby tiny portions of tissue are visually identi fied through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amp lification depended on the type of histologic stain that was used to f acilitate microscopic visualization of the undeparaffinized tissue sec tion. The best results were obtained with methyl green and nuclear fas t red, while Wright's stain yielded less PCR product. Two other stains , Evans blue and light-green SF yellowish (also known as the counterst ain for geomori methenamine silver stain) yielded sufficient PCR produ cts; however, their staining characteristics did not afford satisfacto ry visualization of nuclear chromatin to discriminate between benign a nd malignant cells. Our most significant finding was that a commonly u sed histologic stain, hematoxylin, failed to produce DNA templates tha t could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting materi al for PCR. Among the remaining five strains that were evaluated, the best choice depends on the differential staining characteristics of th e cells to be dissected.