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 formalinfixed 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 (CYCLE min ) and the maximum amount of PCR products (CONC max ). The hematoxylin stain showed a significantly prolonged CYCLE min (P < .01) and lower CONC max (P < .05) in comparison with unstained and untreated control sections. The MayGrunwald stain showed a prolonged CYCLE min (P < .01), although the CONC max 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 CONC max (P < .05), whereas the CYCLE min was not prolonged. The DAB and new fuchsin methods had no untoward effects. Antigenunmasking treatments showed deteriorating effects on PCR. The trypsin treatment significantly prolonged the CYCLE min (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 CYCLE min nor CONC max 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.

Methacarn Fixation for Genomic DNA Analysis in Microdissected, Paraffin-embedded Tissue Specimens

“…Murase et al (2000) reported that immunostaining by visualizing the immunoreactivity with peroxidase-DAB, as in the present study, did not affect PCR efficiency in formalin-fixed PET sections. In the present study, we found substantial loss and degradation of DNA from immunostained sections.…”

“…However, the inhibitory effect of hematoxylin staining on PCR, as observed in large tissue specimens, could be negligible when microdissected small tissue specimens are analyzed (Ehrig et al 2001). We could amplify a 522-bp fragment from 150-270 microdissected cells of HE-stained tissue by 35 cycles of single-step PCR, indicating a superior performance of methacarn fixation for DNA analysis using HE-stained PET specimens to that reported with formalin fixation, by which a 110-bp fragment could be amplified with 38 Ϯ 2.5 PCR cycles using DNA extracted from an unstained tissue section approximately 1 cm in diameter (Murase et al 2000).…”

“…Identification of cells/cellular architecture by nuclear staining is essential for targeted cell microdissection (Burton et al 1998;Murase et al 2000;Serth et al 2000;Ehrig et al 2001). On the other hand, PCR is the major tool for analysis of genomic DNA, and cycle numbers should be minimized to avoid amplification-derived DNA polymerization errors.…”

“…Tissue staining is essential for cell identification in practical molecular analyses using the microdissection technique (Pontén et al 1997;Burton et al 1998;Alcock et al 1999;Fend et al 1999;Murase et al 2000;Serth et al 2000;Gjerdrum et al 2001), and therefore it is important to examine the effect of tissue staining on the performance of molecular analyses as reported in DNA analysis using formalin-fixed PETs (Burton et al 1998;Murase et al 2000;Serth et al 2000). The present study was performed to determine the suitability of methacarn fixation for genomic DNA analysis in terms of target fragment size and number of microdissected cells required, using cresyl violet-stained sections.…”

Quantitative Amplification of Genomic DNA from Histological Tissue Sections after Staining with Nuclear Dyes and Laser Capture Microdissection

“…Previous reports have compared the effect of different histological nuclear dyes on recovery of DNA from formalin-fixed, paraffinembedded tissue samples that were obtained by manual dissection, either by quantitating recovered DNA or examining DNA amplification by polymerase chain reaction (PCR). [2][3][4] These studies concluded that hematoxylin staining resulted in unsatisfactory DNA recovery from the tissue samples, suggesting that hematoxylin would not be a suitable dye for microdissection. The results presented here confirm the unsatisfactory performance of hematoxylin on relatively large tissue fragments sampled manually from histological sections.…”