Induction of 6-thioguanine-resistant lymphocytes in Fischer 344 rats following in vivo exposure to N-ethyl-N-nitrosourea and cyclophosphamide

We have developed a limiting dilution clonal assay for determining the frequency of 6-thioguanine-resistant (TGr) lymphocytes produced in rats by in vivo exposure to genotoxic agents. Spleen lymphocytes were isolated from female Fischer 344 rats and were cultured with 1 microgram/ml of phytohemagglutinin (PHA) for 40 hr. Northern blot analysis revealed that this procedure resulted in increased hprt and beta-actin mRNA synthesis. Conditions for optimum cloning were established by culturing four PHA-primed lymphocytes/well in 96-well round-bottom microtiter plates containing a medium supplemented with interleukin-2. These cultures also contained autologous and/or TK6 feeder cells inactivated with different doses of irradiation. Lymphocyte cloning efficiencies (CEs) were highest in plates containing both irradiated TK6 cells (5 x 10(3) cells/well; 90 Gy) and irradiated autologous feeder cells (5 x 10(4) cells/well; 50 Gy). CE did not depend on the number of primed lymphocytes/well when four or fewer target cells/well were cloned. To measure the effects of chemical mutagens on the frequency of TGr lymphocytes, rats were given a single i.p. injection of 0-150 mg/kg of N-ethyl-N-nitrosourea (ENU), a direct-acting alkylating agent, or 0-50 mg/kg of cyclophosphamide (CP), an indirect acting alkylating agent. Lymphocytes were isolated, primed, and cloned at 4 weeks after CP treatment and at 1, 2, 4 and 6 weeks after ENU treatment. CE in these cultures ranged from 12% to 27%. Cultures were also established for measuring CE in the presence of 6-thioguanine (TG) and these contained 5 x 10(3) irradiated TK6 cells and 5 x 10(4) primed rat lymphocytes/well. The frequency of TGr lymphocytes was calculated by correcting the CE in the presence of TG with the CE measured in its absence. ENU exposure produced a higher frequency of TGr lymphocytes than CP, but both chemicals produced a dose-dependent increase in TGr cells. In addition, the frequency of ENU-induced TGr lymphocytes increased with time after treatment. The TGr cells are presumed to be hprt mutants, but further analysis at the DNA level is required to establish this. The dose-dependent responses obtained with both ENU and CP treatments suggest that rat lymphocytes are sensitive to direct- and indirect-acting alkylating agents administered in vivo and that the rat lymphocyte assay is a useful complement to the in vivo/in vitro mouse assay for determining the mutagenicity of environmental toxicants.     

A simple and rapid immunological technique for visualising chromosome-mediated gene transfer (CMGT)

A method is described for visualising chromosome-mediated gene transfer (CMGT) by detecting chromosomes labelled with bromodeoxyuridine (BrdU) using a monoclonal antibody to BrdU. In this experiment, the CCRF-CEM T cell line was grown in the presence of BrdU and the labelled chromosomes were isolated and transfected into human embryonic fibroblasts. Uptake and retention of chromosomes were compared for transfection with either PEG or DMSO treatments. Following transfection the labelled chromosomes could be visualised in recipient cells using a monoclonal antibody to BrdU, followed by immunoperoxidase staining. Chromosome uptake into cells was similar for both DMSO and PEG treatments and was a relatively frequent event; about 1 in 5 recipient cells had labelled material present. This technique can be used to assess the technical aspects of the earliest stages of chromosome-mediated gene transfer.     

Immunogold cell staining using monoclonal antibodies: application to lymphoid cells on coated slides

The immunogold staining technique was presented for light microscopic detection of cell surface antigens with monoclonal antibodies. The lymphoid cells were adhered to glass slides precoated with the adhesive poly-(dimethyl-diallyl-)ammonium chloride. The staining with the monoclonal mouse antibodies followed by the gold labelled sheep anti-mouse antibody was carried out on the cells adhered to the slides. In spite of the adherence, the patching of the gold marker as the prerequisite for the light microscopic detection occurred and the positively stained cells were visualized. On basis of this method the content of T lymphocytes (BL-T2+), B lymphocytes (BL-Ig-L/1+) and monocytes (BL-M/G+) in the mononuclear cell fraction of peripheral blood was determined in a population of healthy donors and of patients. The content of T lymphocytes of 29 healthy donors was determined using the immunogold staining technique (66.7 +/- 10.4%) and the immunofluorescence technique (67.6 +/- 9.5%). The correlation between the results obtained with both methods was highly significant (p less than 0.001).     

Flash labelling of S-phase cells in short-term organ culture of normal and pathological human endometrium using bromodeoxyuridine and tritiated thymidine

Normal hyperplastic and malignant endometrial specimens were labelled in vitro with bromodeoxyuridine (BrdU). S-phase cells were stained after DNA denaturation, using a monoclonal antibody to BrdU and an indirect immunoperoxidase method. Various conditions for BrdU uptake, DNA denaturation, and staining were tested. BrdU labelling was compared with autoradiography using tritiated thymidine, with good correlation. Glandular labelling indices of proliferative endometrium were significantly higher than both secretory and hyperplastic endometrium but were similar to carcinoma. Stromal labelling showed the same trend but the differences were not statistically significant.     

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine(BrdU) on cell cycle progression and micronucleus inductionwere studied in different mammalian cell cultures. Simultaneousflow cytometric measurements of DNA content and side scatterof nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependenttemporary block in the G₂/M phase of the first cell cycle. NTH3T3 cells and human amniotic fluid fibroblast-like cells, onthe contrary, did not show any cell cycle disturbances in thepresence of BrdU. Micronucleus frequency increased as soon asCHE cells started to divide and reached a plateau when all cellshave divided. The height of this plateau was almost equal for60 and 100 µM BrdU. This saturation of micronucleus inductionwas due to a saturation of BrdU incorporation into DNA alreadyat a dosis of 60 µM as shown by the BrdU/Hoechst quenchingtechnique. Indirect immunofluorescent staining of kinetochoreswith CREST antibodies revealed that nearly all BrdU-inducedmicronuclei were kinetochore-negative suggesting the presenceof acentric chromosome fragments in these micronuclei. DNA distributionsof micronuclei measured by flow cytometry showed several peaksrepresenting micronuclei which contain DNA fragments of definedsizes induced by non-random breakage of chromosomes 1 and Xas verified by flow karyotyping and C-banding.     

Negative cross-reactivity of rabbit anti-Malassezia furfur antibodies with other yeasts

Anti-Malassezia furfur monospecific polyclonal antibodies was produced by repeated immunization of rabbit with Malassezia furfur yeast cells mixed with Freud adjuvant. The antibody titres of respective rabbit's serum samples prior to and after each immunization against M. furfur were assayed by indirect immunofluorescence technique using the M. furfur whole yeast antigen fixed in Teflon coated slides. The highest anti-M. furfur antibody titre achieved was 1 in 1280 dilution. At 1:20 dilution, none of the respective serum samples taken at various stages of immunization gave positive immunofluorescent staining against any of the other species of yeasts tested in this study. Anti-M. furfur monospecific polyclonal antibodies produced in rabbit in this study has the potential for diagnostic application in immunohistochemical detection of M. furfur in human tissues.     

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.     

Quantitative and rapid assays of togaviruses by immunofluorescence.

For the quantitative assay of selected togaviruses, suspensions of BHK cells were inoculated with virus and grown in spinner cultures. At intervals, dependent on the growth characteristics of the viruses, about 10(3) cells were centrifuged on to microscope slides and then stained with fluorescent antibody. For rapid demonstration (2--3 hr) of specific viral antigens, virus was bound in successive dilutions onto microscope slides and stained. Binding of specific anti-virus antibodies in both methods, was determined either by a second labelled antibody against complement or by labelled protein A. By these methods the determination of viral antigens (Sindbis, Semliki Forest, West Nile and hog cholera viruses) was independent from the source of immune sera.     

Transforming growth factor-beta 1 knockout mice. A mutation in one cytokine gene causes a dramatic inflammatory disease.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.     

Drug-resistant lymphocytes in man as indicators of somatic cell mutation

Direct in vivo tests of somatic mutation in man may provide realism in assessing the genetic risks of potential environmental mutagens. The autoradiographic determination of purine analogue (8-azaguanine; 6-thioguanine) resistant (AGr; TGr) peripheral blood lymphocytes (PBLs) arising in vivo in man is proposed as a candidate test. PBLs bearing the naturally occurring Lesch-Nyhan (LN) mutation are prototype mutant cells. LN PBLs are AGr and TGr, whereas normal PBLs are AG and TG sensitive. When judged by the inhibition of phytohemagglutinin (PHA) stimulated 3H-thymidine incorporation in vitro, analogue-resistant LN PBLs may be distinguished from analogue-sensitive normal PBLs by several methods. Early studies quantitating PHA stimulation by scintillation spectrometry detected down to 1% of LN PBLs in artificial mixtures with normal PBLs. Although LN heterozygous females could be identified on the basis of lymphocyte mosaicism, scintillation spectrometry was too insensitive to detect rare "LN-like" PBLs in non-LN individuals. Autoradiography, however, detected rare TGr PBLs in normal non-LN individuals. Their frequencies did not increase with age. With this method, TGr PBL frequencies in LN heterozygous females were found to range from 1 x 10(-3) to 5 x 10(-2), whereas blood samples from LN males showed from 23% to 100% TGr cells. Rare LN PBLs could be detected in artificial mixtures with normal cells. Studies in human patients undergoing various potential mutagenic therapies assessed the effects of these therapies on the TGr PBL variant frequencies (Vf) of non-LN individuals. Group TGr PBL Vf values were higher in treated patient groups than in controls. However, some untreated patient groups (cancer and psoriasis) also had elevated values, suggesting that disease itself may affect TGr PBL frequencies. Nonetheless, one patient group (vitiligo) showed elevated Vf values in treated (8-methoxypsoralen and long-range UV light = PUVA) but not in untreated patients, suggesting that treatment was responsible for the TGr PBL elevations. Longitudinal studies over time in cancer patients receiving X-irradiation therapy demonstrated that such exposures also are associated with TGr PBL frequency rises and suggested that longitudinal studies may be necessary to relate TGr PBL Vf elevations to specific environmental influences. Variant TGr PBLs were found at frequencies comparable to those in man in the peripheral blood of rats. They increased in a single study following treatment of the animals with a clinical alkylating agent. Characterization of the TGr PBLs suggests that some of these cells are mutants. Presumably the mutant cells arise in vivo by somatic cell mutation.     

Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.     

Immunoelectron microscopic localization of thyroglobulin in the human thyroid gland

The microidentification of the organelles containing thyroglobulin (TG) in the follicular cells of human thyroid glands were studied by the immunoelectron microscopic method. Fresh human thyroid glands were used for the purification of TG. TG was purified by differential salt fractionation and DEAE-cellulose chromatography. Anti-human TG rabbit IgG antibody was obtained by immunization of this purified TG. F(ab')2 fragments of anti-TG rabbit IgG antibody were prepared by pepsin digestion. The specificity of the antibody was tested on an Ouchterlony immunodiffusion plate. Conjugation of the purified F(ab')2 fragments of anti-TG rabbit IgG antibody to horse radish peroxidase was performed in order to use the direct peroxidase labelled antibody method. Under immunohistochemical light microscopy, the luminal colloid and the follicular cells appeared heavily stained. Under immunoelectron microscopy, positive reactions were observed in the rough endoplasmic reticulum, the Golgi apparatus, and the apical cell border. To date, no clear direct evidence of the presence of TG in the rough endoplasmic reticulum and the Golgi apparatus of human thyroid cells has ever been reported in immunoelectron microscopic studies. This study indicates that the use of F(ab')2 fragments of anti-TG rabbit IgG antibody in immunoelectron microscopic study is useful for the identification of TG in organelles of human thyroid follicular cells.     

One heat-induced antigen retrieval step allows the sequential detection of two antigens on the same slide of tissue fixed and enclosed in paraffin

The heat-induced antigen retrieval (HIAR) procedure allows the immunohistochemical detection of various antigens on paraffin-embedded sections. The re-use of slides negative for the detection of a first set of antigens may be an interesting alternative in case of a limited number of slides. After HIAR, a series of Bouin's liquid-fixed tonsil sections was stained for Epithelial Membrane Antigen (EMA) which labelled epithelial cells and plasma cells. A second immunostaining for CD20 (L26) was performed on the same slides divided in two sets. The HIAR was repeated in the first set but not in the second one. A similar staining of follicular B-cells was observed in the two sets. However background staining was enhanced by repetition of HIAR. Analogous results were obtained using anti-cytokeratin (KL1) instead of anti-EMA. This was confirmed on slides for which three or four cycles of HIAR were performed prior immunostaining. Our data suggest that the renewal of the HIAR procedure must be avoided since it was found stable for at least 1 year.     

Immunohistochemical phenotype of malignant mesothelioma: predictive value of CA125 and HBME-1 expression

Histological diagnosis of malignant mesothelioma and differentiation from adenocarcinoma is often difficult. Definitive pathological confirmation of malignant mesothelioma requires demonstration of an appropriate immunohistochemical phenotype. Selection of an optimum panel of immunohistochemical antibodies for the reliable identification of malignant mesothelioma is hindered by the absence of a specific immunohistochemical label for mesothelioma cells. Recently, we have found that the ovarian carcinoma cell antibody CA125 labels malignant mesothelioma cells, and the antibody HBME-1 has been developed as a sensitive mesothelial cell marker. We have compared the immunohistochemical staining patterns achieved with CA125 and HBME-1 to those obtained using a panel of eight further antibodies in 17 malignant mesotheliomas and 14 primary and secondary adenocarcinomas within lung and pleura. CA125 labelled malignant mesothelioma cells in 15 of 17 cases (88%), and adenocarcinoma cells in seven of 14 cases (50%). HBME-1 labelled mesothelioma cells in all 17 cases (100%) but also labelled adenocarcinoma cells in 10 of 14 cases (71%). BerEP4 positively labelled one malignant mesothelioma but was negative in the remaining 16 cases and positively labelled nine of 14 adenocarcinomas (64%). Monoclonal anti-CEA, AUA-1, CA19.9 and LeuM1 labelled no malignant mesotheliomas and were positive in 10 (71%), nine (64%), eight (57%) and six (43%) of 14 cases of adenocarcinoma, respectively. Diastase-PAS staining detected neutral mucin in none of the malignant mesotheliomas but in 10 (71%) of the 14 adenocarcinomas. We conclude that CA125 and HBME-1 do not label mesothelial cells with sufficient specificity to be useful for differentiating malignant mesothelioma from adenocarcinoma, although negative staining with HBME-1 makes a diagnosis of malignant mesothelioma unlikely. As there remains an absence of a specific positive mesothelial cell marker this distinction is still most reliably made using a panel of antibodies including at least two of the following: anti-CEA, AUA-1, BerEP4, LeuM1 and CA19.9, in combination with histochemical assessment of neutral mucin production.     

Detection of apoptoses in gastro-intestinal graft-versus-host disease and cytomegalovirus colitis by a commercially available antibody against caspase-3

The common pathway of any apoptotic cascade leads to the activation of the so-called execution caspases, particularly caspase-3 (CSP3). The question of whether immunohistochemical (IHC) detection of activated CSP3 might be useful for routine purposes still needs to be clarified. We analyzed apoptoses in gastrointestinal graft-versus-host disease (GvHD) and cytomegalovirus (CMV) colitis using a commercially available polyclonal antibody against activated human CSP3. In GvHD samples obtained from the colon, apoptoses detected by CSP3 varied between 11 and 43/40 crypts, and in esophageal specimens between 21 and 40/1.5 mm squamous epithelium basal length. This count was correlated with the apoptotic count assessed on hematoxylin- and eosin (H&E)-stained slides. A perfect concordance for those counting between 30 and 40 apoptoses/40 crypts or 1.5 mm squamous epithelium basal length was detected, whereas cases with low apoptotic counts on the H&E stained slides showed a 2 to 3 fold greater number of stained nuclei as assessed by CSP3 staining. In CMV-colitis, although the number of exploding crypt cells was 8-13/40 crypts, only 1-2 nuclei/40 crypts and almost all cells with typical nuclear inclusions stained positively. The presence of CMV can be easily detected on H&E- or IHC-stained slides, while masked GvHD by an overlying CMV-colitis might remain unrecognized. Staining for CSP3 may be helpful in distinguishing these two conditions, as apoptotic count would be excessive in GvHD.     

Proliferative characteristics and sister chromatid exchange (SCE) of colony-forming cells (CFU-S) in acute myelogenous leukemia

Sister chromatid exchange (SCE) and cell cycle progression of colony-forming cells (CFU-S) from patients with acute myelogenous leukemia were studied using bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. The mean SCE rate of CFU-S was 7.99, which is similar to that reported for human peripheral blood lymphocytes. Cultures treated with BrdU at culture initiation and then harvested 72-120 hr later indicated a cell cycle time (TC) of approximately 60 hr. However, cells allowed to proliferate for 72-120 hr prior to the addition of BrdU showed TC values of 12-36 hr. Analysis of serial cultures from one patient revealed a heterogeneous population with a mean TC of 19.5 hr. These cell cycle times are considerably shorter than previously published reports and suggest that leukemic cells have the potential to divide rapidly.     

Noscapine hydrochloride-induced numerical aberrations in cultured human lymphocytes: a comparison of FISH detection methods and multiple end-points

The cytokinesis block in vitro micronucleus (MN) assay in combination with CREST staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows mechanistic information on the induction of numerical chromosomal aberrations to be obtained through a rapid and simple microscopic analysis. These techniques can now be used to investigate relationships between the induction of chromosomal loss, non-disjunction and polyploidy by aneuploidy-inducing agents. In the present study, we treated 72 h cultured lymphocytes for the last 24 h of culture with various concentrations of the cough medicine noscapine hydrochloride (NOS) (3.9-120 micro g/ml) in the presence of either cytochalasin B (CYB) (3 micro g/ml) or 5-bromo-2'-deoxyuridine (BrdU) (1 micro M). Using the CREST staining modified MN assay in the CYB-treated cultures, we detected significant increases in CREST-positive but not CREST-negative MN in both binucleated and, to a lesser extent, mononucleated cells, demonstrating the ability of this compound to induce chromosomal loss. In addition, using FISH with chromosome 1- and 9-specific classical satellite probes, a significant induction of chromosomal non-disjunction in the binucleated lymphocytes and polyploidy in the mononucleated lymphocytes was seen, indicating that polyploidy induced by NOS may occur without progression through a normal anaphase and/or telophase. In the BrdU-treated cultures, a dose-dependent induction of hypodiploidy, hyperdiploidy and polyploidy was observed using FISH with a chromosome 9-specific alpha-satellite probe in the labeled cells. By comparison, in the unlabeled non-cycling cells, only a slight increase in hyperdiploidy/polyploidy but not hypodiploidy was seen. A comparison of the effects seen at different concentrations shows that at the lower effective concentrations, all three types of numerical aberrations, chromosomal loss, non-disjunction and polyploidy, contributed to the numerical aberrations seen, whereas at the highest concentration tested, polyploidy was the predominant alteration. These studies indicate that FISH in combination with CYB or BrdU immunfluorescent staining can be sensitive tools for the identification of aneuploidy-inducing agents.     

Measurement of the S-phase duration in normal and abnormal human endometrium by in vitro double labelling with bromodeoxyuridine and tritiated thymidine

The duration of the S phase of the cell cycle (ts) in the glands and stroma of normal, hyperplastic, and malignant endometrium was measured in vitro using a double labelling technique. Endometrial organ cultures were incubated sequentially in tritiated thymidine (3HTdR) and bromodeoxyuridine (BrdU) each for 1 h. Labelled cells were visualized in tissue sections using 3HTdR autoradiography and immunohistochemistry with an anti-(BrdU) antibody. Three populations of cells, single labelled with either 3HTdR or BrdU, and double labelled cells, were easily identified and ts was calculated from both the rate of influx into, and efflux out of S. There were no significant differences in ts in the different tissue types or between influx and efflux in the glands. However, in the stroma ts calculated from the rate of efflux was significantly shorter than that calculated from the rate of influx, suggesting that during the cell culture period the rate of cell efflux from S was greater than influx. This is the opposite to what would be expected in an exponentially growing cell population and suggests that there may be an effect from the culturing conditions.     

Chick embryo proliferation studies using EdU labeling

Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S‐phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The antibody against BrdU is also large and tissue penetration can be difficult. EdU (5′‐ethynyl‐2′‐deoxyuridine) is closely chemically related to BrdU, with detection achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. Cell cultures, flow cytometry and high throughput studies using EdU‐labeled cells is exceptionally fast and does not require denaturation or antibodies. We have developed a tissue‐labeling technique in chick embryos using EdU. Following EdU chemistry to detect proliferating cells, the tissue can undergo immunolabeling. We demonstrate fluorescent EdU chemistry followed by Tuj1 antibody staining resulting in multiplex fluorescent tissues. Developmental Dynamics 238:944–949, 2009. © 2009 Wiley‐Liss, Inc.     

抗DESMOPLAKIN I单克隆抗体的制备

作者从水牛鼻表皮中分离桥粒,提取桥粒中的Desmoplakin I(DPI)。用DPI作免疫原,免疫BALB/C小鼠,建立了一株分泌抗DPI单抗的细胞AD-1。免疫组化染色证实,该单抗与上皮组织呈阳性反应,与非上皮性组织呈阴性反应。提示该单抗可作为区分上皮性肿瘤与非上皮性肿瘤的免疫组化探针。