Role of hyaluronan and CD44 in reactive oxygen species-induced mucus hypersecretion

5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 μM for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC.     

The effects of social environment on adult neurogenesis in the female prairie vole

In the mammalian brain, adult neurogenesis has been found to occur primarily in the subventricular zone (SVZ) and dentate gyrus of the hippocampus (DG) and to be influenced by both exogenous and endogenous factors. In the present study, we examined the effects of male exposure or social isolation on neurogenesis in adult female prairie voles (Microtus ochrogaster). Newly proliferated cells labeled by a cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), were found in the SVZ and DG, as well as in other brain areas, such as the amygdala, hypothalamus, neocortex, and caudate/putamen. Two days of male exposure significantly increased the number of BrdU-labeled cells in the amygdala and hypothalamus in comparison to social isolation. Three weeks later, group differences in BrdU labeling generally persisted in the amygdala, whereas in the hypothalamus, the male-exposed animals had more BrdU-labeled cells than did the female-exposed animals. In the SVZ, 2 days of social isolation increased the number of BrdU-labeled cells compared to female exposure, but this difference was no longer present 3 weeks later. We have also found that the vast majority of the BrdU-labeled cells contained a neuronal marker, indicating neuronal phenotypes. Finally, group differences in the number of cells undergoing apoptosis were subtle and did not seem to account for the observed differences in BrdU labeling. Together, our data indicate that social environment affects neuron proliferation in a stimulus- and site-specific manner in adult female prairie voles.     

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.     

Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo

This protocol describes methods for calculating the proliferative parameters of cell populations. The basis of the technique is to label cells, either in vitro or in vivo, with halogenated thymidine analogs, such as bromodeoxyuridine (BrdU). Bivariate DNA-BrdU flow cytometry is used to analyze the BrdU-labeled and unlabeled cells. The enumeration of specific cohorts of cells that either have or have not divided in the interval between labeling and cell/tissue sampling permits the calculation of the potential doubling time (T(pot)) of the population, plus the durations of DNA synthesis (T(S)) and the G2+M phase (T(G2+M)) of the cell cycle. The method provides information that is not otherwise available, namely inhibition of DNA synthesis and the separate evaluation of cell-cycle effects in BrdU-labeled and unlabeled subpopulations. Ethanol-fixed samples take 1 d to prepare and stain, and reliable parameter estimates might be obtained from measurements made at a single time point after labeling.     

Comparison of protein A-gold and ferritin immunoelectron microscopy of Semliki Forest virus in mouse brain using a rapid processing technique

Processing tissue for transmission electron microscopy by standard laboratory methods can take two to three days. This makes the development of new techniques time consuming and generally restricts the use of the electron microscope in routine diagnostic work. The possibility of viewing tissue with the electron microscope five hours after sampling using rapid processing techniques is presented. The morphology of the tissue appears undamaged with cell and organelle ultrastructures being readily recognized, as is the presence of virus and its replicating stages. When combined with immunoelectron microscopy a rapid labeling protocol is possible. We have used the technique to develop protein A-gold (6 and 16 nm particles) and ferritin immunoelectron microscopic techniques to demonstrate viral antigens in brain cell cultures and brain tissue from mice infected with Semliki Forest virus.     

A stereologic approach to estimate the number of immunogold-labeled molecules in cells of tubules

OBJECTIVE: The quantity of molecules can be measured very precisely by molecular biological methods, but the capabilities of these are limited to measure only the total mass of tissue. For estimating the number of molecules at the cell level, it is necessary to combine an immunohistochemical protocol with designed-based principles of stereology at the level of electron microscopy (EM). This article focuses on the problems and practical solutions of fitting together immunohistochemistry, stereology, and electron microscopy for the estimation of the number of angiotensin II AT1 receptors in rat kidney arterioles. STUDY DESIGN: We performed the preembedding immunostaining of angiotensin II AT1 receptors using the silver-enhanced immunogold labeling system at EM level on serial sections of renal arterioles from 5 rats. RESULTS: Using this method the number of molecules can be estimated along the renal arterioles separately on the cell's surface, in cytoplasm, in nucleus, or in any subcellular location. CONCLUSION: For estimating the number of AT1 receptors, we designed a protocol that took into account the requirements for both immuno-EM and stereology. This method can be applied for estimating any molecule number in different types of cells in tubules.     

In Situ Hybridization at the Electron Microscopic Level Using a Bromodeoxyuridine Labeled DNA Probe

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM). and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.     

Two-color Fluorescence Labeling in Acrolein-fixed Brain Tissue

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-μm sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling. (J Histochem Cytochem 58:359–368, 2010)     

Improved biocytin labeling and neuronal 3D reconstruction

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.     

Regulation of parietal cell migration by gastrin in the mouse

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5'-bromo-2'-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10-30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.     

Imaging activation of adult-generated granule cells in spatial memory

New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes the immunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice. To label adult-generated granule cells, mice are injected with the proliferation marker 5-bromo-2'-deoxyuridine (BrdU). At different delays after BrdU treatment, mice are trained to locate a hidden platform in the Morris water maze, and spatial memory can then be tested in a probe test with the platform removed from the pool. Ninety minutes after this probe test, mice are perfused and tissue is sectioned. Immunohistochemical procedures are used to quantify BrdU-labeled cells and expression of the immediate early gene, Fos. Because Fos expression is regulated by neuronal activity, the degree of overlap between BrdU-labeled and Fos-labeled neurons provides an indication of whether adult-generated granule neurons have been incorporated into spatial memory circuits.     

Detection of in situ mitotic activity of dendritic epidermal T-cells by BrdU labeling.

We analyzed mitotic dendritic epidermal T-cells (DETC) in the epidermis of C3H/He (Thy-1.2+) mice, using double immunoenzymatic labeling. Ear skin was incubated with 100 microM bromodeoxyuridine (BrdU) for 5 hr and then either directly studied or cultured for an additional 12 hr in BrdU-free medium. After BrdU labeling, with or without additional culture, epidermal sheets were obtained by ethylenediaminetetraacetic acid separation. The epidermal specimens were immunostained by the peroxidase method to visualize nuclear BrdU and then by the biotin-streptavidin-alkaline phosphatase method for surface Thy-1.2 antigen. In specimens processed immediately after BrdU labeling, a mean 3.0% of all basal cells were labeled with BrdU and a mean 1.1% of the BrdU-labeled cells were also positive for Thy-1.2. Moreover, a mean 2.1% of the DETC had incorporated BrdU. BrdU-labeled DETC had a variety of appearances; they were dendritic and round in the BrdU-treated specimens, while oval and paired cells were also found in the specimens after additional culture. These morphological changes of BrdU-labeled DETC demonstrate that resident DETC can become mother cells undergoing mitosis through the retraction of their dendrites, and it appears that DETC divide at a relatively high rate, i.e., up to 10% of the DETC may enter the S-phase of the cell cycle every 24 hr.     

Early radiation-induced endothelial cell loss and blood-spinal cord barrier breakdown in the rat spinal cord

Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood-brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood-spinal cord barrier permeability was assessed using immunohistochemistry for albumin and (99m)Tc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood-spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood-spinal cord barrier disruption in the rat spinal cord after irradiation.     

Induction of mammotroph development by a combination of epidermal growth factor,insulin, and estradiol-17beta in rat pituitary tumor GH3 cells

Several reports have indicated that prolactin-secreting cells (PRL cells) are generated from growth hormone-secreting cells (GH cells). We have shown that treatment with a combination of epidermal growth factor (EGF), insulin, and estradiol-17beta (E (2)) induces the appearance of PRL cells in pituitary tumor GH3 cells. The aim of the present study was to clarify the involvement of mitosis in the cytogenesis of PRL cells in rat pituitary and GH3 cells. The effects of the treatment with EGF, insulin and E(2) on DNA-replication were studied by detecting the uptake of bromodeoxyuridine (BrdU) into the nucleus. In cultured rat pituitary cells, BrdU-labeled PRL cells were observed irrespective of the hormone treatment. In GH3 cells, BrdU-labeled GH cells and mammosomatotrophs (MS cells) were detected; BrdU-labeled PRL cells were not detected, however, when GH3 cells were treated with BrdU for 3 hr and then immediately examined for BrdU-labeling. BrdU-labeled PRL cells were found only when GH3 cells treated with BrdU were allowed to grow for another 3 days. This finding suggests that during the additional 3-day culture, BrdU-labeled PRL cells were generated from BrdU-labeled cells other than PRL cells. These results indicate that PRL cells are transdifferentiated from GH cells or MS cells in GH3 cells by a combined treatment with EGF, insulin and E(2), while PRL cells in rat pituitaries are able to proliferate in response to the hormone treatment. Thus, there may be two pathways for cytogenesis of PRL cells: the transdifferentiation of GH cells or MS cells, and a self-duplication of PRL cells.     

Genome-wide analysis of replication timing in mammalian cells: Troubleshooting problems encountered when comparing different cell types

DNA is replicated in a defined temporal order that is developmentally regulated and constitutes a unique and stable fingerprint of a given cell type. Recently, we developed a robust assay to profile replication timing genome wide that can be applied to essentially any proliferating cell population. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU). The cells are sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated (BrdU IP), amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray (or sequenced). Since the basic steps of this protocol have been detailed elsewhere, here we focus on problems encountered when adapting this protocol to different cell types or tissue sources and modifications that have been successfully applied to troubleshoot these problems. There is an increasing demand for such studies to address how replication is regulated during development, its relationship to chromatin architecture and other chromosome functions, and the relevance of cell culture models to regulation in the native organismal niche.     

自体骨髓单核细胞移植在肝纤维化兔肝脏内的定植能力

目的建立四氯化碳诱导的兔肝纤维化动物模型,观察体外分离标记的自体骨髓单核细胞（ABM-MNCs）经肠系膜上静脉自体移植至肝纤维化区及周边区后的存活、定植状况。方法将40只普通级日本大耳家兔随机分为细胞移植组和对照组各20只,实验组腹腔注射40%CCl4橄榄油溶液建立肝纤维化模型,对照组腹腔注射等量生理盐水。细胞移植组于模型稳定后自体髂骨处抽取骨髓,采用氯化氨红细胞溶解法分离得到单核细胞,以5溴-2脱氧尿嘧啶核苷（BrdU）标记体外ABM-MNCs及鉴定;分离培养ABM-MNCs,将3×10^9个ABM-MNCs经肠系膜上静脉回输体内,对照组回输等量生理盐水,移植前、移植后3、7、14、21 d分别取肝组织固定,进行免疫组织化学检测。结果BrdU体外标记ABM-MNCs的免疫组织化学表现示：20μmol/L BrdU孵育ABM-MNCs 72 h的阳性标记率达95%;肝组织20μmol/L BrdU免疫组化染色切片显示：自体骨髓单核细胞移植后第3天,肝小叶中央静脉周围BrdU染色阳性,随着时间的推移,阳性染色逐渐增强,并逐步向肝组织内部延伸。阳性染色主要分布于肝组织汇管区周围组织,而对照组BrdU染色则阴性。结论ABM-MNCs经肠系膜上静脉移植后,可在纤维化区及周边区存活,定植。     

Effect of fixation and epitope retrieval on BrdU indices in mammary carcinomas.

Studies in which 5-bromo-2'-deoxyuridine (BrdU) is used to quantify rates of cell proliferation are conducted prospectively. Therefore, the opportunity exists to select conditions that optimize detection of the BrdU epitope. The objective of this study was to quantify the extent to which the BrdU epitope was masked by formalin vs methacarn fixation in the assessment of cell proliferation. Mammary carcinomas from animals pulse-labeled with BrdU were trisected. A portion was frozen and the remaining two portions were fixed in 10% neutral buffered formalin or methacarn for 24 hr, processed, embedded in paraffin, and sections stained for incorporated BrdU using a peroxidase immunohistochemical staining technique. Antigen retrieval techniques also were applied to formalin-fixed sections. Fixation in methacarn gave the highest labeling index (16.4%), which was comparable to that observed in unfixed frozen sections (17.5%). Formalin fixation alone dramatically suppressed the labeling index (0.3%), which was only partially recovered using various antigen retrieval techniques (2.1-8.1%). Methacarn fixation is recommended for prospective studies in which BrdU detection is planned because of the quantitative recovery of epitope and the simplicity of the approach.     

Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots

The importance of locating proteins in their context within cells has been heightened recently by the accomplishments in molecular structure and systems biology. Although light microscopy (LM) has been extensively used for mapping protein localization, many studies require the additional resolution of the electron microscope. Here we report the application of small nanocrystals (Quantum dots; QDs) to specifically and efficiently label multiple distinct endogenous proteins. QDs are both fluorescent and electron dense, facilitating their use for correlated microscopic analysis. Furthermore, QDs can be discriminated optically by their emission wavelength and physically by size, making them invaluable for multilabeling analysis. We developed pre-embedding labeling criteria using QDs that allows optimization at the light level, before continuing with electron microscopy (EM). We provide examples of double and triple immunolabeling using light, electron and correlated microscopy in rat cells and mouse tissue. We conclude that QDs aid precise high-throughput determination of protein distribution.     

Pinocytosis as a select marker of ramified microglia in vivo and in vitro

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.