Effect of fixation time and microwave oven heating time on retrieval of the Ki-67 antigen from paraffin-embedded tissue.

Abstract

Microwave oven heating has been employed for retrieval of antigens from formalin-fued, p a d h e m b e d d e d tissue for immunohistochemical staining. Recently, a Ki-67 equivalent murine monoclonal antibody was generated which can detect tumor proliferative activity in routinely processed tissue with microwave oven heating. We assessed the effect of fixation time (4, 24, or 48 hr) and microwave oven heating time (7, 14, 21,28, 35, or 49 min) on retrieval of the Ki-67antigen from tonsil tissue. Ki-67 staining was quantitated by image analysis. Owing to the heterogeneity of Ki-67 staining within and between germinal centers, we employed a measurement technique that averages staining across the ger- IntroductionTumor proliferative activity has prognostic value in many solid tumors (1-4). Immunohistochemical analysis of tumor proliferation has been accomplished with the Ki-67 monoclonal antibody (MAb) which recognizes a nuclear antigen expressed throughout the cell cycle ( 5 ) . Unfortunately, this MAb cannot be used for retrospective studies that employ formalin-fixed, paraffin-embedded tissue. Recently, however, a Ki-67 equivalent murine MAb was generated which can detect tumor proliferative activity in routinely processed tissue (6,7). Immunohistological detection with this antibody requires antigen retrieval by microwave oven heating according to the method described by Shi et al. (8).The mechanism of action of microwave oven heating for retrieval of antigens is poorly understood. Shi et al. (8) hypothesized that the cross-linking effect of formaldehyde fixation is altered by microwave heating. They reported that tissues fixed in formalin for as long as 2 years could be stained for pancytokeratin and vimentin using antigen retrieval. However, the effect of overfixation could not be quantitatively determined because optimally fixed tissue SM is a Visiting Scientist from the Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan. from the same anatomic site was not available. In addition, the relationship between formalin fixation time and microwave oven heating time was not examined. We describe here the effect of fixation time and microwave oven heating time on retrieval of the Ki-67 antigen. We employed a quantitative approach with image cytometry (9,lO). Materials and MethodsTissue and Fixation. Fresh human tonsil tissue was obtained from routine surgical specimens. Tissue was cut into three equal pieces of approximately 4 mm3 and immediately fixed in 10% neutral buffered formalin for 4, 24, or 48 hr. After fixation the tissue was immersed in 70% ethanol until processing. All tissue were processed simultaneously. Fixed tissues were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks, which were cooled before sectioning. Four-pm thick sections were cut and mounted on 3-aminopropyltriethoxysilane (APES) (Sigma; St Louis, M 0 ) -coated slides. The use of a tissue adhesive helped to prevent section detachment during microwave processing. Sec...

Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.

“…Lucassen and A. Williams, unpublished results), indicating that for perfusion-fixed tissue a minima1 duration of post-fixation is required to render MW pre-treatment before ISEL effective. Although human and rat tissue differ with respect to antemortem factors, our data indicate that immersion-fixed postmortem tissue does not benefit from MW pre-treatments before ISEL, which differs from MW application in immunocytochemistry, which was shown to be successful even after short fixation times (Iwamura et al, 1994;Munakata and Hendricks, 1993). This difference might be explained by differences in temperature sensitivity and tissue distribution between proteins and DNA.…”

Histopathological Analyses of Proliferative Potentials of Intracranial Meningiomas Using Bromodeoxyuridine and MIB-1 Immunohistochemistry

“…Usually antigen retrieval of Ki-67 is carried out in MIB-1 immunohistochemistry. Munakata and Hendricks stated that microwave oven heating for at least 14 min yielded significant antigen retrieval of Ki-67 in MIB-1 immunohistochemistry using routinely processed paraffin sections [38]. Recent researchers have carried out sufficient antigen retrieval in MIB-1 immunohistochemistry.…”

Antigen Retrieval Immunohistochemistry: Past, Present, and Future

“…In fact, the results of immunostaining on routinely processed tissue using AR are usually equal to (and occasionally better than) the frozen tissue. Several studies have suggested that increased heating may reverse the effects of longer fixation (Munakata and Hendricks 1993).…”