Anti nestin antibody

BBMD3 was synthesized by reacting natural BBM with NH₂-containing substitutents. The structure and purity of the products were analyzed using HNMR spectroscopy. BBMD3 displayed over 98% purity by NMR analysis. Human miRNA inhibitors (synthetic oligonucleotides) against hsa-miRNA-4284 and has-miRNA-27a were purchased from GeneCopoeia. RNAiFect Transfection reagent was purchased from Qiagen Inc. Recombinant human epidermal growth factor (EGF) and fibroblast growth factor basic (FGF) were obtained from R&D Systems Inc., and Accutase was purchased from Innovative Cell Technologies. B-27 serum-free supplement (50X) for growth and maintenance of neurons was obtained from Gibco. Heparin (1000 units/ml) was purchased from APP Pharmaceuticals, LLC. Horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were purchased from GE Healthcare. Anti-nestin antibody was purchased from EMD Millipore. All other antibodies were purchased from Cell Signaling, Inc.

5-Ethynyl-2′-deoxyuridine (EdU) (Click-iT EdU Alexa Fluor 488 Imaging Kit, Invitrogen) is a nucleoside analog of thymidine. It is incorporated into DNA during active DNA synthesis. Cell proliferation was determined by the EdU incorporation assay, as previously described10 (link). Briefly, 100 μL of 2× working solution of EdU was added to the wells with FNSCs in the 96-well plate during the exposure time for 6 hours with different sevoflurane concentrations. After incubation, the cells were fixed with 4% paraformaldehyde for 15 min and then 0.2% Triton X-100 for 20 min at room temperature. The cells in each well were washed twice with 3% BSA between every two steps. Then, 100 μL of Click-iT reaction cocktail was added to each well for 30 min and protected from light. The cells were blocked with 3% BSA for 1 h and then incubated with the anti-nestin antibody (Millipore; 1:500 dilution) overnight at 4 °C. After washing with the 3% BSA, the cells were incubated with the secondary antibody Alexa Fluor 555 for 1 h and then with the 1× Hoechst 33342 solution for 10 min in the dark at room temperature. Each well was washed twice with PBS, and the images were obtained using an Olympus IX-70 inverted fluorescence microscope(Olympus America Inc, Center Valley, PA).

Esophageal carcinoma specimens were collected from patients diagnosed at the Department of Pathology of the First Affiliated Hospital of Xinxiang Medical University. The study was approved by the Ethical Committee of Xinxiang Medical University, and conformed to the Declaration of Helsinki and the Good Clinical Practice Guidelines. All patients participating in this project provided written informed consent.
Dulbecco’s modified eagle media (DMEM) and Fetal bovine serum (FBS) were purchased from GIBCO. The anti-nestin antibody was purchased from Millipore and anti-GAPDH antibody was purchased from Santa Cruz Biotechnology.

Cells were fixed with 4% paraformaldehyde (Wako) for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 10% goat serum (Nichirei Bioscience) in PBS for 1 hr at room temperature (RT). Cells were incubated with primary antibodies for 2 hr at RT before incubation with appropriate secondary antibodies for 1 hr at RT, prior to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI)(Dojindo Laboratories). Samples were observed using a Leica fluorescent microscope (Leica Microsystems). The primary antibodies used were: anti-nestin antibody (1:100; Millipore), anti-fibronectin antibody (1:100; Sigma-Aldrich), anti-αSMA antibody (1:100; Sigma-Aldrich), anti-S100β antibody (1:100; Sigma-Aldrich) and anti-trichohyalin antibody (1:5; Santa Cruz Biotechnology). Secondary antibodies used were: Alexa Fluor 488-conjugated goat anti-mouse (1:200) and Alexa Fluor 546-conjugated goat anti-rabbit (1:200, both from Life Technologies).

For immuno-peroxidase staining, fixed cells were permeabilized using Triton X 0.1% (Sigma Aldrich) for 15 min in PBS, rinsed in PBS to facilitate antigen retrieval, and then incubated in 3% hydrogen peroxide (H₂O₂) for 20 min to block endogenous peroxidase activity, followed by treatment with a blocking solution containing 10% normal goat serum in PBS for 1 h at room temperature. Then, cells were incubated overnight at 4 °C, with the primary antibody diluted in PBS containing 2% normal goat serum. The following primary antibodies were used: mouse monoclonal anti-α-syn antibody (1:1000; Abcam, Cambridge, UK; Table 1) and rabbit polyclonal anti-nestin antibody (1:1000; Millipore, Temecula, CA, USA). The secondary antibody was an anti-mouse or anti-rabbit biotin-conjugated antibody (1:200; Vector Laboratories, Burlingame, CA, USA), and the reaction was carried out for 1 h at room temperature. Cells were exposed to the avidin–biotin complex (ABC, Vector Laboratories) for 1 h. Then, they were exposed for 3 min to the peroxidase substrate diaminobenzidine (DAB, Vector Laboratories), at room temperature. After dehydration in increasing alcohol solutions, cells were clarified in xylene, cover-slipped with DPX mounting medium (Sigma Aldrich), and observed under a Nikon Eclipse 80i light microscope (20× magnification).

Cells were fixed with 4% paraformaldehyde (Sigma) for 30 min at room temperature. The cells were then incubated with the primary antibodies including anti-Nestin antibody, anti-MAP2 antibody, and anti-GFAP antibody (1:1000, Millipore, Temecula, CA, USA) in phosphate-buffered saline (PBS), with 0.1% Triton X-100 (Sigma-Aldrich) and 2% normal goat serum (Vector Laboratories, Burlingame, CA, USA) overnight at 4 °C. After rinsing with PBS three times, the cells were incubated with the secondary antibodies at room temperature for 3 h. The cells were then incubated with 1 μg/mL 4′,6-diamidino-2-phenylindole (Roche, Switzerland) for 15 min to stain the nuclei. After rinsing with PBS, fluorescence mounting medium (Dako, Aglient, Santa Clara, CA, USA) was added, and immunoreactivity images were obtained and processed under a confocal microscope (FluoView FV1000, Olympus, Tokyo, Japan).