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  • Cytogenesis in the adult monkey motor cortex: perivascular NG2 cells are the major adult born cell type. 25308320

    We used confocal microscopy and immunohistochemistry (IHC) to look for new cells in the motor cortex of adult macaque monkeys that might form the cellular bases of improved brain function from exercise. Twenty-four female Macaca fascicularis monkeys divided into groups by age (10-12 years, 15-17 years), postexercise survival periods, and controls, received 10 weekly injections of the thymidine analog, bromodeoxyuridine (BrdU) to mark new cells. Sixteen monkeys survived 15 weeks (5 weeks postexercise) and 8 monkeys survived 27 weeks (12 weeks postexercise) after initial BrdU injections. Additionally, five Macaca mulatta female monkeys (∼5.5-7 years) received single injections of BrdU and survived 2 days, 2 weeks, and 6 weeks after BrdU injections. Neural and glial antibodies were used to identify new cell phenotypes and to look for changes in proportions of these cells with respect to time and experimental conditions. No BrdU(+) /DCx(+) cells were found but about 7.5% of new cells were calretinin-positive (Cr(+) ). BrdU(+) /GABA(+) (gamma-aminobutyric acid) cells were also found but no new Cr(+) or GABA(+) cells colabeled with a mature neuron marker, NeuN or chondroitin sulfate antibody, NG2. The proportion of new cells that were NG2(+) was about 85% for short and long survival monkeys of which two, newly described perivascular phenotypes (Pldv and Elu) and a small percentage of pericytes (2.5%) comprised 44% and 51% of the new NG2(+) cells, respectively. Proportions of NG2(+) phenotypes were affected by post-BrdU survival periods, monkey age, and possibly a postexercise sedentary period but no direct effect of exercise was found.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5320
    Nombre del producto:
    Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody

  • Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies. 15314088

    Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Development of a highly sensitive immunohistochemical method to detect neurochemical molecules in formalin-fixed and paraffin-embedded tissues from autopsied human brains ... 25784860

    Immunohistochemistry (IHC) is a valuable method for identifying discrete neurochemical molecules by the interaction of target antigens with validated antibodies tagged with a visible label (e.g., peroxidase). We have developed an immunostaining method that is highly sensitive in detection of neurochemical antigens. Our IHC method, which we call the PBTA method, involves a hybrid protocol that implements aspects of both the polymer and avidin-biotin-complex (ABC) methods in combination with biotin-tyramide amplification. When using [Met]-enkephalin as a target antigen, the sensitivity of the PBTA method for IHC was more than 100-fold higher compared with the polymer and ABC methods. In addition, its sensitivity for enzyme-linked immunosorbent assay was about 1,000-fold higher compared with the ABC method. We examined the utility of our IHC method for both chromogenic and fluorescence detection systems used to visualize neurochemical peptides and proteins in formalin-fixed, paraffin-embedded tissues from autopsied human brains. The results convincingly demonstrate that under optimal conditions, our IHC method is highly sensitive without increasing non-specific background activities. Our IHC method could be a powerful tool for detection and visualization of neurochemical antigens that are present even in trace amounts in autopsied human brains.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5026

  • Identification and validation of genes involved in cervical tumourigenesis. 21338529

    Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer.A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay.Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells.Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3861

  • Leptin targets in the mouse brain. 19350671

    The central actions of leptin are essential for homeostatic control of adipose tissue mass, glucose metabolism, and many autonomic and neuroendocrine systems. In the brain, leptin acts on numerous different cell types via the long-form leptin receptor (LepRb) to elicit its effects. The precise identification of leptin's cellular targets is fundamental to understanding the mechanism of its pleiotropic central actions. We have systematically characterized LepRb distribution in the mouse brain using in situ hybridization in wildtype mice as well as by EYFP immunoreactivity in a novel LepRb-IRES-Cre EYFP reporter mouse line showing high levels of LepRb mRNA/EYFP coexpression. We found substantial LepRb mRNA and EYFP expression in hypothalamic and extrahypothalamic sites described before, including the dorsomedial nucleus of the hypothalamus, ventral premammillary nucleus, ventral tegmental area, parabrachial nucleus, and the dorsal vagal complex. Expression in insular cortex, lateral septal nucleus, medial preoptic area, rostral linear nucleus, and in the Edinger-Westphal nucleus was also observed and had been previously unreported. The LepRb-IRES-Cre reporter line was used to chemically characterize a population of leptin receptor-expressing neurons in the midbrain. Tyrosine hydroxylase and Cre reporter were found to be coexpressed in the ventral tegmental area and in other midbrain dopaminergic neurons. Lastly, the LepRb-IRES-Cre reporter line was used to map the extent of peripheral leptin sensing by central nervous system (CNS) LepRb neurons. Thus, we provide data supporting the use of the LepRb-IRES-Cre line for the assessment of the anatomic and functional characteristics of neurons expressing leptin receptor.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1542
    Nombre del producto:
    Anti-Tyrosine Hydroxylase Antibody