The DAB staining was performed using a commercial kit called DAB Detection Kit (EliVision Super DAB, Maixin biotech company, Fuzhou, China). After paraffin sections of vaginal tissues as well as 3D cultures solvent-dewaxing and treatment with microwave antigen retrieval, endogenic peroxidase blocker were added at room temperature for 10 mins and the primary antibodies mouse anti-p63 (1:100, ab735, Abcam, Cambridge, UK), rabbit anti-Laminin (1:100, ab11575, Abcam, Cambridge, UK), mouse anti-CK18 (1:100, sc-32329, Santa Cruz Biotechnology, CA, USA), mouse anti-CK14 (1:100, sc-23878, Santa Cruz Biotechnology, CA, USA), mouse anti-E-cadherin (1:100, sc-71008, Santa Cruz Biotechnology, CA, USA), mouse anti-Dsg-1 (1:100, sc-13716, Santa Cruz Biotechnology, CA, USA) were respectively incubated on the slides at room temperature for an hour. The slides were then added the reaction-amplified reagent for 20 mins and conjugated with high-sensibility enzyme-conjugated lgG polymer. Reactants were visualized with the fresh-prepared DAB chromogenic solutions for 3 to 5 mins. Hematoxylin somatic cell staining reagent was used to counterstain nuclei for 8 mins. Glass slides were finally mounted with neutral balsam and visualized under EVOS visual imaging microscope (Life Technologies Corp Bothell, WA, USA).
Sc 71008
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-71008 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is a device intended for use in scientific research applications. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab735, by Abcam (1 mentions)
Ab11575, by Abcam (1 mentions)
Evos visual imaging microscope, by Thermo Fisher Scientific (1 mentions)
Dab detection kit, by Maixin Group (1 mentions)
Mouse anti ck14, by Santa Cruz Biotechnology (1 mentions)
Mouse anti ck18, by Santa Cruz Biotechnology (1 mentions)
Sc 32329, by Santa Cruz Biotechnology (1 mentions)
Mouse anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Sc 23878, by Santa Cruz Biotechnology (1 mentions)
Ab9021, by Abcam (1 mentions)
Forskolin, by Merck Group (1 mentions)
Ab58968, by Abcam (1 mentions)
Du 530, by Beckman Coulter (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Genegnome imaging system, by Syngene (1 mentions)
4 protocols using sc 71008
1
Immunohistochemical Analysis of Vaginal Tissue
2
Isolation and Culture of Cytotrophoblasts
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The isolation and culture of CTBs from human term placentae were performed as previously described (Wang et al., 2014 (link)). More than 95% of the CTBs harvested stained positive for cytokeratin 7 (CK7) (ab9021, RRID: AB_306947, Abcam), a marker for CTBs. CTBs that spontaneously fused to form syncytia in vitro were identified by immunostaining for E-cadherin (sc-71008, RRID: AB_1121199, Santa Cruz Biotechnology, USA; 3195, RRID: AB_2291471, Cell Signaling Technology). The human choriocarcinoma cell line BeWo was obtained from the American Type Culture Collection. BeWo cells were cultured and treated with forskolin (F6886, Sigma-Aldrich) to induce cell–cell fusion as described previously (Wang et al., 2014 (link)). The resultant lentiviruses encoding EGFP (control), TTL, mutated TTL, VASH2, TTL shRNA, or shNM (control) were used to infect BeWo cells to establish stable cell lines (BeWoGFP, BeWoTTL, BeWoTTLmut, BeWoVASH2, BeWoshTTL, and BeWoshNM, respectively). The fluorescence-positive cells were sorted by fluorescence-activated cell sorting and cultured and maintained in Ham’s F-12K (Kaighn’s)/DMEM (1:1) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin.
3
Western Blot Analysis of Cell Lysates
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The cells were washed three times with cold PBS and mixed with whole-cell lysis buffer (4 mM EGTA, 3 mM EDTA pH 8.0, 125 mM NaF, 0.5 mM NA 3 VO 4 , 2.5 mg/ml aprotinin, 25 mg/ml trypsin inhibitor, 12.5 mM HEPES pH 7.4, 1% Triton X-100, and 25 mM phenylmethylsulphonyl fluoride) as described previously (Zhang et al. 2013) (link). A BCA Protein Assay Kit (Pierce Biotechnology, Thermo Scientific, Dubuque, IA, USA) was used to determine the protein concentration by spectrophotometry at 562 nm (Beckman DU530, Fullerton, CA, USA). Twenty-five micrograms of each protein sample was subjected to western blotting analysis with the following primary antibodies: rabbit polyclonal anti-nephrin (1:500; ab58968, Abcam, Cambridge, MA, USA), rabbit polyclonal anti-human b-hCG (1:1000; ab54410, Abcam), mouse monoclonal anti-E-cadherin (1:1000; sc71008, Santa Cruz Biotechnology), mouse monoclonal anti-bactin (1:2000; TA-09, Zhongshan Golden Bridge Crop., Beijing, China), rabbit polyclonal anti-syncytin 2 (1:500; AP13018A, Abgent, San Diego, CA, USA), mouse monoclonal anti-connexin 43 (1:1000; 610061, Transduction Laboratories, Lexington, KY, USA), and HRP-conjugated secondary antibodies. Signals were detected by the GeneGnome Imaging System (Syngene Bio-imaging, Cambridge, UK).
4
Immunofluorescence Staining for E-cadherin
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The immunofluorescence experiments were performed as previously described (Wang et al. 2014 (link), Li et al. 2015) (link). Briefly, cells were fixed, washed and stained with mouse monoclonal E-cadherin antibody (sc-71008, Santa Cruz Biotechnology).
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