Nuclei, isolated from paraffh-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the fomalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffinembedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices @I) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P < 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P < 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.
A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in (1) increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and (2) In cell kinetic research, there is a growing interest in tured cells, human bone marrow cells, and tumor xenothe use of immunocytochemistry for the detection of 5-grafts in n d n u mice. Because of its high sensitivity, its bromodeoxyuridine (BrdUrd), which S-phase cells have high cell yield from solid tumors, and its low backincorporated into their DNA (6,9-12). Bivariate flow ground staining, this method is especially suitable for cytometric analysis of BrdUrd incorporation and DNA the analysis of tumor cells labeled in vivo. content allows the calculation of cell kinetic parameters, such as phase distribution, growth fraction, and cell MATERIALS AND METHODScycle traverse time. The most important advantage of Several cell lines were used for this study, including this method is that it obviates the need for radioactive MCF7 (breast cancer cells), 5583 (16), DNA precursors and the time-consuming methods for and 2246 (colorectal carcinoma cells). The cell lines 5583 their detection (17). and 2246 are a gift from Dr. K. Verstijnen (University The immunocytochemical method requires in situ de-of Limburg, Department of Pathology). All cell lines naturation of DNA in order to make the incorporated were grown at 37°C in Dulbecco's modified medium BrdUrd accessible to the antibody. This has mainly been supplemented with 10% fetal calf serum (FCS). Human accomplished by applying either acid (2,4) or heat (3). bone marrow was collected freshly. After lysis of red Acid denaturation results in a low BrdUrd detection blood cells in 0.155 M NH&l, white blood cells were level (3), which may be insufficient for the detection of cultured at 37°C in RPMI 1640, supplemented with 10% slowly cycling cells in solid tumors after in vivo admin-FCS. istration of BrdUrd. In contrast, thermal denaturationBrdUrd Labeling of cellular DNA shows higher sensitivity (31, but cannot be applied to cells from bone marrow or peripheral blood, Cell lines and human bone marrow cells were pulse because they are more fragile than other cells and there-labeled in vitro by incubation (30 min) in BrdUrd confore mostly destroyed during thermal denaturation.taining medium (10 pM final concentration). In one exHere we describe a new method for the preparation of periment cells were cultured in the presence of BrdUrd nuclei for immunocvtochemical detection of BrdUrd. using pepsin digestion of ethanol fixed cells or tissue, followed by acid denaturation of the obtained nuclei. This method is applicable to a variety Of Cells, including CUl-Address reprint requests to B. Schutte,
We investigated the influence of various fixatives on monoclonal anti-BrdUrd antibody binding of BrdUrd-substituted DNA in tissue sections of routinely processed mouse small intestine after in vivo administration of BrdUrd. For denaturing fixatives such as ethanol or Carnoy's fluid, a standard denaturation protocol showed specific crypt cell labeling. With cross-linking agents such as formalin and glutaraldehyde, a remarkable increase in staining intensity was obtained after tissue digestion with pepsin before acid denaturation. The optimal pepsin concentration was determined for maximal immunoreactivity combined with acceptable morphology.
We developed a rapid and convenient immunocytochemical method for simuftaneous detection ofantigen expression and S-phase cells by means ofanti-bromodeoxyuridine (BrdUrd) antibodies. Immunocytochemical detection of BrdUrd after in vivo administration in mice was compared with autoradiography using [3H]-BrdUrd. Both the sensitivity and specificity of the technique were high. For the dual peroxidase staining technique, DAB color modification by cobalt ions was used. We showed that antigen localization was not af-Recently, non-autoradiographic methods for distinguishing DNA-synthesizing cells have been developed. These use monocbonal antibodies to measure incorporation ofbromodeoxyuridine (BrdUrd) into cell DNA (7-14). By use of immunocytochemical detection methods, the number and localization ofcells containing BrdUrdsubstituted DNA can be determined. Combination of this techniquc in double labeling experiments with immunocytochemical staining
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.