Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

Summary The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [³H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and ³H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the ³H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

BrdUrd incorporation studies for evaluation of spermatogenesis in the blue fox.

The utility of BrdUrd incorporation techniques for studies of spermatogenesis was investigated in the blue fox. BrdUrd was injected intraperitoneally followed by collection of testicular tissue by castration/hemicastration at intervals up to 35 days after pulse labelling. Fluorescent tagged monoclonal antibodies against BrdUrd allowed detection of cells with incorporated tracer in histological sections by fluorescent light microscopy as well as in isolated testicular cells by bivariate BrdUrd/DNA flow cytometry. The duration of the spermatogenic cycle was estimated by following the labelled cohort of preleptotene spermatocytes by immunofluorescence in sections through the various stages of maturation to the late spermatid stage. These data were confirmed by bivariate BrdUrd/DNA flow cytometry of testicular cells in suspensions. Furthermore, estimations of the S phase durations and length of the spermatogonial cell cycle were possible. A consistent and satisfactory fluorescence intensity of incorporated label throughout the study shows that degradation of the incorporated label is no practical problem for this type of study, and suggests that the method is an excellent tool for studying aspects of proliferation and maturation during normal as well as perturbed spermatogenesis. Advantages of the described method include avoidance of potential radiation influence on spermatogenesis from commonly used radiolabelled tracers, e.g., 3H-TdR, and that both large and small animals can be investigated at modest cost since the unlabelled BrdUrd is considerably less expensive than labelled tracers.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

RADIOTOXICITY OF INCORPORATED [3H]THYMIDINE AS STUDIED BY AUTORADIOGRAPHY AND FLOW CYTOMETRY CONSEQUENCES FOR THE INTERPRETATION OF FLM DATA

The radiotoxic effects of incorporated [³H]thymidine on proliferation kinetics of flash-labelled (30 min, 0.3 μCi/ml, 40 Ci/mM) L-929 cells in vitro were studied by means of autoradiography and flow cytometry. the flow cytometric results obtained by applying the BUdR-33258 Hoechst technique, using new evaluation procedures, showed that the labelled cells are delayed in their progression through the S and G₂+ M phases, leading to mitotic delay. From autoradiographs, the fraction of labelled mitoses was determined and, in addition, the ratio of labelled and of unlabelled mitotic cells to all cells. the radiotoxic effects are not evident from the FLM curve, even if the ratio of labelled mitotic cells to all cells shows a highly distorted shape. A mathematical model has been developed that describes the perturbed cell kinetics due to radiotoxic effects of the incorporated [³H]thymidine. These findings have considerable consequences for the interpretation of autoradiograhic data, especially of labelled mitoses curves.     

Proliferative capacity of embryonic chick notochord: a comparaison between PCNA positivity versus 3H-thymidine incorporation.

The proliferation rate of the embryonic chick notochord is measured during its functional period. Chick embryos of 4 different age groups respectively, 3.5, 4.5, 5.5 and 6.5 days of development, are selected. To measure proliferation activity in the notochords, two different methods are used. 1. Notochords are immunostained for proliferating cell nuclear antigen (PCNA). 2. After in vivo incorporation of ³H-thymidine (³H-TdR), DNA synthesis is visualized by autoradiography. After embedding in paraffin, 5 μm sections are cut and stained with PC10 monoclonal antibody and avidine biotin peroxidase complex (ABC) technique. Corresponding chick embryos of the same age groups are injected in ovo with ³H-thymidine. After fixation 2 μm sections are made and exposed for autoradiography. Results obtained by PCNA and autoradiography labelling techniques for the evaluation of the proliferative capacity are different. Both however show a regression of the proliferative activity. The regression of the number of cells that went through S-phase, as shown by autoradiography, is lower than that of PCNA positive cells. So, a number of notochordal cells stops in G1 phase. Our results moreover, indicate that the proliferating activity of the notochord of chick embryos is higher at young developmental stages than at later stages.     

The proliferation of adrenal medullary cells in newborn and adult mice

Summary The proliferative activity of newborn and adult mouse adrenal medullary cells was determined with light and electron microscopic autoradiography. The H³ thymidine labelling index of 2 weeks old mice adrenal medullary cells was about 9.4 % and declined to less than 1 % in adult mice. In electron microscopic autoradiography labelled norepinephrine as well as epinephrine cells could be seen. Only in 1 and 2 weeks old mice some morphologically undifferentiated cells were visible. In formaldehyde induced fluorescence combined with light microscopic autoradiography the fluorescence intensities of labelled and unlabelled medullary cells were measured. On average the fluorescence intensity of labelled cells was lower than that of unlabelled cells. The differences could be explained by a higher number of autoradiographic silver grains laying on the cytoplasm of labelled cells. These results give evidence that fully differentiated adrenal medullary cells are capable of division.This study was supported by Jubiläumsfonds der Österreichischen Nationalbank grant No. 818     

Does ex vivo labelling of proliferating cells in colonic and vaginal mucosa reflect the S-phase fraction in vivo?

The ex vivo labelling of DNA-synthesizing epithelial cells in colonic and vaginal mucosa was compared with in vivo labelling. For this purpose, in vivo S-phase cells were labelled with [3H]thymidine (Tdr) and ex vivo labelling was continued by culturing tissue specimens in bromodeoxyuridine (BrdU). Various methods of tissue culture were employed in order to improve diffusion of medium (and BrdU) in the tissue. BrdU and 3H-TdR labelling were evaluated by immunohistochemistry and autoradiography respectively. Ex vivo labelling resulted in a patchy distribution of labelled cells, which did not correspond with the 3H-TdR labelling pattern obtained in vivo. Under the described conditions ex vivo labelling does not appear to be a reliable for estimation of the proliferative activities in vivo.     

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([³H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase-anti-alkaline-phosphatase technique, the peroxidase-anti-perox-idase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G₂+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Summary Bromodeoxyuridine (BrdUrd) immunohisto-chemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and ³H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0:05% proteinase at 37°C for 20 min and hydrolysis with 1 N HCl at 37°C for 20 min was sufficient to detect BrdUrd immunore-activity in ³H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd, immunohistochemistry in cell-kinetic studies.     

Moderate and severe malnutrition alters proliferation of spleen cells in rats

Objectives

Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo effects of moderate and severe malnutrition on spleen cell proliferation in 21‐day‐old rat pups.

Materials and methods

Spleen cell proliferation was determined following administration of bromodeoxyuridine (BrdUrd) over a time course of 2, 4, 6 and 8 h. Incorporation of BrdUrd was detected using FITC‐conjugated anti‐BrdUrd monoclonal antibodies and total DNA content was detected and evaluated using propidium iodide using flow cytometry.

Results

Proportions of cells in S and G₂/M were reduced in the rats with moderate (MN2^nd) and severe (MN3^rd) malnutrition. BrdUrd incorporation was lower in both groups of malnourished rat. In cells of MN2nd individuals, length of G₁ became shorter, while length of S‐phase increased. In contrast, fraction of cells in proliferation was significantly lower in both groups of malnourished rat, with MN3rd group having lowest percentage of cell population growth. In this study, severe malnutrition did not significantly affect duration of phases of the cell cycle, although fractions of proliferating cells were dramatically reduced.

Conclusion

Moderate malnutrition increased time of cells in DNA synthesis and time of total cell cycle and severe malnutrition reduced growth fraction of spleen cells in malnourished rats during lactation.

     

Sister chromatid induction by β-irradiation from incorporated 3H-thymidine: a paradox explained

Sister chromatid exchanges (SCE's) induced by [3H]thymidine (³HdT) of increasing specific activities incorporated over one cycle and 5-bromodeoxyuridine (BrdUrd) over the two following cycles were investigated in synchronised Chinese hamster ovary (CHO) cells. SCEs induced during the first cycle on a T.T template (SCE 1) show little increase with dose compared with those induced in the second cycle on a ³HT.T template (SCE 2) where the linear increase with dose reflects that seen after X irradiation. During the third cycle, SCEs 3.1 and 3.2 are induced on unlabelled T.B or labelled ³HT.B templates respectively. These templates are theoretically present in a 11 ratio after random segregation at second metaphase. Over practically the entire dose range however, the ratio 3.1/3.2, which dereased with dose, was >1.0 and similar to the high values obtained by other workers. At increasing times after BrdUrd introduction, the ratio decreased from >1.0 to <1.0. Measurements showed that the expected 50% level of labelled chromosomes at metaphase in the samples could vary between 42%–59%. Cells with >50% labelled chromosomes were more delayed in the cell cycle due to the ³H-irradiation than those with <50%. Early fixations therefore favoured SCE 3.1 while late favoured SCE 3.2. SCEs due to BrdUrd in ³HT.B and T.B templates showed no synergistic interaction with irradiation-induced SCEs. When these BrdUrd-induced SCEs were removed from the totals then the ³H-induced SCE levels in ³HT.T, and ³HT.B templates (SCE 2 and 3.2) were similar and increased at a similar rate with dose. This was 2–3 times faster than in SCE 1 and 3.1 where the SCE levels due to irradiation were again similar but lower than for 2 and 3.2. The -irradiation source is therefore most effective in inducing SCEs when present in the replicating fork and considerably less effective when it is just behind the fork (SCE 1) and/or in the surrounding chromosomes in the cell.     

Electron microscopic autoradiographic and electron microscopic immunohistochemical studies on the anti-HPO antibody-producing cells

The secondary immune responses in mouse popliteal lymph nodes to horseradish peroxidase (HPO) were studied by a combination of electron microscopic autoradiography and electron microscopic immunohistochemistry in order to clarify the relationship between antibody-producing and DNA-synthesizing capacities of the plasmacytic series. The anti-HPO antibody-containing cells, which increased in number 72 h after the secondary antigenic stimulation, were mainly immunoblasts and immature plasma cells. Immunoblasts containing anti-HPO antibody incorporated [³H]thymidine more actively than did immature plasma cells containing anti-HPO antibody. In 144 h after the secondary antigenic stimulation, antibody containing cells consisted mainly of mature plasma cells and immature plasma cells. Immature plasma cells containing the anti-HPO antibody incorporated a little [³H]thymidine, but mature plasma cells containing anti-HPO antibody did not incorporate any [³H]thymidine.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

Comparison of isotopic and immunohistochemical methods of studying epithelial cell proliferation in hamster tongue

Our purpose was to validate different approaches to the study of cell proliferation in stratified squamous epithelia, using oral mucosa as a model. Dorsal and ventral tongue from the hamster were examined following in vivo labelling with tritiated thymidine and bromodeoxyuridine (BrdUrd), and in vitro labelling with BrdUrd. These were compared with direct immunolabelling of fixed tissue sections with monoclonal antibody PC10. For the former methods S phase cells were quantified following autoradiography or immunohistochemistry. We conclude that the proliferative status of simple, flat, lining mucosae such as ventral tongue can be derived by all three prelabelling methods and, on average, 18–19 cells per surface millimetre length were in DNA synthesis. On the other hand dorsal tongue epithelium, which is thicker, has an undulating morphology and a complex cell renewal pattern, gives different results with the three labelling methods. In both sites the proliferating cell nuclear antigen (PCNA) index was fourfold that obtained by nucleotide labelling. This is consistent with PCNA marking proliferative cells in other phases of the cell cycle in addition to the S phase. Thus, there are potential differences between the information on proliferative status derived by PCNA immunohistochemistry and other established cell cycle markers, which need to be taken into account in the interpretation of epithelial cell kinetic data in health and disease.     

Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis

Abstract We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2–3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (<2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found that the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.     

Bromodeoxyuridine Immunohistochemistry for Evaluating Age-Related Changes in the Rat Mandibular Condyle Decalcified by Intravenous Infusion

Age-related changes in cell proliferation kinetics of the mandibular condyle were studied in Sprague-Dawley rats using bromodeoxyuridine (BrdUrd) immunohistochemistry in decalcified, paraffin-embedded tissues. Intraperitoneal injection of 40 mg/kg BrdUrd was given 1 hr before animal sacrifice. Continuous perfusion of EDTA solution via the left ventricle shortened the decalcification time. Immunohistochemical staining was performed with monoclonal anti-BrdUrd antibody. BrdUrd labeled cells, i.e., the S-phase cells, were clearly visible with well-preserved cytological detail. Their nuclei exhibited homogeneously stained granules. The labeling index in the intermediate zone of the condyle decreased with increasing age of the animals. This method is useful for evaluating physiological and pathological changes of the rat mandibular condyle as well as other bones and joints.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

Bromodeoxyuridine and lododeoxyuridine Double Immunostaining for Epoxy Resin Sections

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.