Influence of Histochemical and Immunohistochemical Stains on Polymerase Chain Reaction

Abstract

The polymerase chain reaction (PCR) analysis of DNA extracted from tissue sections can be applied to a variety of research and diagnostic protocols. To analyze selectively the specific areas of tissue, a direct microdissection of histochemically or immunohistochemically stained sections, if satisfactory for PCR, is helpful. However, the influence of various staining methods on PCR has been poorly investigated. In this study, paraffin sections of formalin-fixed lymph node samples were histochemically stained with Mayer's hematoxylin, eosin Y, methyl green, or May-Grunwald solution and immunostained for CD45 using 3,3′-diaminobenzidine (DAB), DAB with cobalt ion (DAB-Co), or new fuchsin as the chromogen. In addition, unstained sections were treated with trypsin, microwave, or pressure cooker, the techniques frequently used in immunostains for antigen unmasking. DNA was extracted from each section, and the PCR efficiency in amplifying a 110 bp portion of the beta-globin gene was evaluated by two parameters: the cycle count in which the first visible band was obtained (CYCLEmin) and the maximum amount of PCR products (CONCmax). The hematoxylin stain showed a significantly prolonged CYCLEmin (P <.01) and lower CONCmax (P <.05) in comparison with unstained and untreated control sections. The May-Grunwald stain showed a prolonged CYCLEmin (P <.01), although the CONCmax was not significantly different from that of the control (P =.051). The eosin and methyl green stains showed no effects against PCR. In immunostains, the DAB-Co method showed a lower CONCmax (P <.05), whereas the CYCLEmin was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigen-unmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLEmin (P <.01), and the PCR amplification did not reach the “plateau” level with a maximum of 60 cycles. The PCR efficiency was worse in microwave or pressure cooker treatment, with neither CYCLEmin nor CONCmax being obtained. When target areas from sections for subsequent PCR amplification are microdissected, methyl green is most suitable as a dye for nuclear staining. The immunohistochemical visualization with DAB or new fuchsin yields no unfavorable effects. A successful PCR amplification may not be expected in sections that are pretreated in a microwave oven or pressure cooker.