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Localization of Connective Tissue Growth Factor: RESULTS(1)

Mar 20, 2013 Author: Walter Mcneil | Filed under: Embryo

RESULTS(1)Localization of CTGF in the Mouse Uterus during the Estrous Cycle

We have previously demonstrated the use of affinity-purified anti-mCTGF for the specific immunohisto-chemical detection of CTGF in mouse and human fibroblasts. Using this antibody, CTGF was localized in the nonpregnant mouse uterus to the luminal and glandular epithelial cells (Fig. 1, A-C). Staining was distributed throughout the entire cell and was not preferentially localized to either the apical or basal surface. In contrast, stromal cells and the myometrium showed low or undetectable levels of CTGF. Epithelial staining for CTGF was more intense during the early proestrous (Fig. 1A) and diestrous (Fig. 1B) stages than at estrus (Fig. 1C). Sections stained with preimmune IgG were negative for CTGF at all stages, for which a typical example is shown in Figure 1D. Staining with the CTGF antibody was reduced to negative control levels by prior incubation of the antibody with a 1000fold excess of mCTGF peptide antigen (data not shown). ampicillin antibiotic

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  • Localization of Connective Tissue Growth Factor: MATERIALS AND METHODS(4)

    Mar 19, 2013 Author: Walter Mcneil | Filed under: Embryo

    Heparin-Affinity Chromatography

    Mouse ULF collected from 16 animals were pooled and subjected to heparin-affinity fast protein liquid chromatography (FPLC) using a TSK heparin 5PW column. Briefly, the column was developed with a 40-ml gradient of 0.2-2 M NaCl in 10 mM Tris-HCl (pH 7.4), and the eluate was collected into forty 1-ml fractions. Fractions were assayed at 50 ^l/ml for their ability to stimulate Balb/c 3T3 cell DNA synthesis as measured by [3H]thymidine incorporation. CTGF was subsequently detected in the fractions by Western blotting.

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  • Localization of Connective Tissue Growth Factor: MATERIALS AND METHODS(3)

    Mar 18, 2013 Author: Walter Mcneil | Filed under: Embryo

    MATERIALS AND METHODS(3)

    Immunohistochemistry

    Uteri were fixed in Histochoice for 5-6 h and then left in 70% ethanol overnight. They were processed in a TP 1050 pressure-vacuum processor (Leica, Deerfield, IL) and embedded in paraffin. Sections (5 ^m) were cut using a microtome and placed on Superfrost Plus slides. The slides were heated at 60°C for 1 h and deparaffinized in xylene for 5 min. Sections were then hydrated by being passed through a series of graded ethanol washes of 95%, 90%, 70%, and 50% ethanol and finally being placed in distilled water for a few minutes. The sections were then treated with 3% hydrogen peroxide to destroy any endogenous peroxidase activity, and then washed in PBS. birth control yasmin

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  • Localization of Connective Tissue Growth Factor: MATERIALS AND METHODS(2)

    Mar 17, 2013 Author: Walter Mcneil | Filed under: Embryo

    To generate pregnant mice, the females were housed with male mice of the same species at around 2000 h. Females were checked for vaginal plugs the next morning, and only those that showed a plug were considered pregnant. This time point was defined as Day 0.5 of pregnancy. Nonpregnant females or mice pregnant at the indicated day were killed and the uteri removed and fixed. A total of 42 animals were used in this study: 21 staged mice for immunohistochemistry (diestrous, n = 2; proestrous, n = 3; estrous n = 2; and pregnant Days 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5, n = 2, 2, 2, 3, 1, and 2, respectively) and 21 nonstaged cycling mice for analysis of CTGF in endometrial extracts (n = 5) or uterine luminal flushings (ULF) (n = 16). ULF were obtained by sequential passage of 1 ml PBS through each uterine horn of groups of either 6 or 10 mice. Aliquots of each ULF sample were tested for their stimulation of DNA synthesis in quiescent cultures of Balb/c 3T3 cells and also subjected directly to SDS-PAGE and Western blotting. buy flovent inhaler

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  • Localization of Connective Tissue Growth Factor: MATERIALS AND METHODS(1)

    Mar 16, 2013 Author: Walter Mcneil | Filed under: Embryo

    MATERIALS AND METHODS(1)

    Materials

    Histochoice was obtained from Amresco (Solon, OH). Superfrost Plus Slides and Permount were purchased from Fisher Scientific (Pittsburgh, PA). Hydrogen peroxide was from Sigma Chemical Company (St. Louis, MO). PBS was obtained from Gibco BRL (Gaithersburg, MD). Normal goat serum (NGS) was from Vector Labs. (Burlingame, CA). Biotin-conjugated goat anti-rabbit IgG (preadsorbed to rat and mouse tissue), peroxidase-conjugated streptavidin and liquid diaminobenzidine-concentrated substrate pack were purchased from BioGenex (San Ramon, CA). Hematoxylin was obtained from Newcomer Supply (Middleton, WI). A TSK heparin 5PW column (0.8 X 7.5 cm) was obtained from TosoHaas (Philadelphia, PA). Heparin Se-pharose was from Amersham Pharmacia Biotech (Piscata-way, NJ). CTGF antisera raised against residues 80-93 or 246-259 of mouse CTGF were produced as previously described. Anti-mCTGF was affinity purified and used for immunohistochemistry and Western blotting. Anti-mCTGF was used for Western blotting. buy ventolin inhalers

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  • CTGF is encoded by a TGFp-in-ducible immediate early gene, although other growth factors such as basic FGF, EGF, and platelet-derived growth factor also induce CTGF expression, albeit to a lesser extent. To date, most studies of the role of CTGF in vivo have focused on the function of CTGF in wound healing and fibrotic disorders, especially those in which TGFp appears to be important. However, a role for CTGF in normal uterine physiology has been inferred from the presence of its mRNA in cycling and early-pregnant pig endometrium. In addition, immuno-precipitation experiments demonstrated synthesis by pig endometrial explants of the 38-kDa CTGF protein, which appears to undergo limited proteolysis yielding bioactive 10- to 20-kDa moieties that are readily detectable as the principal isoforms of CTGF in pig uterine secretory fluids. buy cipro

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  • Mouse(1)

    The last decade has seen the identification of polypeptide growth factors and cytokines as mediators of many of the growth-promoting properties of steroid hormones as well as components of maternal-embryo signaling at the implantation site. Rodent models have produced a plethora of data from which molecules such as leukemia inhibitory factor, colony stimulating factor, epidermal growth factor (EGF), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factors a and (3 (TCFa, TGFp), and insulin-like growth factors have been strongly implicated in regulating uterine remodeling, implantation, and placenta-tion. The collective and coordinate action of these molecules on uterine and extraembryonic cells is likely to be a major mechanism whereby pregnancy is successfully established and maintained. cialis professional

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