Brain tissues and cells were lysed for total protein extraction using SDS buffer. Protein concentrations were determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories) according to the manufacturer’s protocol. Equal amounts of protein were subjected to Western blotting analysis. Antibodies included those specific for Aβ (ab201060, Abcam, Cambridge Science Park, Cambridge, UK), PEDF (MAB1059, Millipore, Boston, MA), PS1 (ab76083, Abcam), phosphorylated c-Jun N-terminal kinase (P-JNK; #3270S, Cell Signaling Technology, Boston, MA), JNK (ab119944, Abcam), BACE1 (#5606S, Cell Signaling Technology), APP (#2452S, Cell Signaling Technology), low-density lipoprotein receptor-related protein 6 (LRP6; SC-25317, Santa Cruz, CA), phosphorylated extracellular signal-regulated kinase (P-ERK; SC-7383, Santa Cruz), ERK (SC-94, Santa Cruz), p38 mitogen-activated protein kinase (MAPK; #9211S, Cell Signaling Technology), P-p38 MAPK (#9212S, Cell Signaling Technology), and β-actin (A544, 2 ml, Sigma).
Mab1059
Manufactured by Merck Group
Sourced in Denmark
MAB1059 is a monoclonal antibody designed for use in laboratory research applications. It is produced using standard hybridoma technology. The core function of MAB1059 is to serve as a specific detection and capture reagent for target analytes in various immunoassay formats.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Ab201060, by Abcam (2 mentions)
Ab76083, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ab119944, by Abcam (1 mentions)
P p38 mapk, by Cell Signaling Technology (1 mentions)
Bace1, by Cell Signaling Technology (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ecl western blotting detection kit, by GE Healthcare (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Rat monoclonal anti vegf antibody, by BioLegend (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Imagequant 400, by GE Healthcare (1 mentions)
Ponceau s red, by Merck Group (1 mentions)
Slide flask, by Thermo Fisher Scientific (1 mentions)
7 protocols using mab1059
1
Protein Expression Analysis in Brain Tissue
2
Brain Tissue Fixation and Immunofluorescence
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Brain tissues were removed after perfusion to flush away the blood and then fixed in ice-cold 4% paraformaldehyde overnight. Then, the tissues were dehydrated in a gradient of sucrose solutions (10%, 20%, 30%) overnight before tissue sectioning. For cell immunofluorescence, cells were plated on culture slides and then fixed in ice-cold 4% paraformaldehyde for 30 min. Then, the samples were prepared according to the protocol as described previously [26 (link)]. The slices were visualized under a confocal microscope (Axio Observer Z1, ZEISS, Jena, Germany). Antibodies included those specific for Aβ (ab201060, Abcam), PEDF (MAB1059, Millipore), PS1 (ab76083, Abcam), and Renilla luciferase (ab185926, Abcam).
3
Western Blot Analysis of VEGF and PEDF
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Five micrograms of eye homogenates samples was mixed with Laemmli buffer (Bio-Rad, CA, USA), boiled for 5 min, separated on 10%–12% SDS-PAGE gels, and transferred to a nitrocellulose membrane. After blocking with 5% skimmed milk (w/v), 0.1% Tween 20 (w/v) in TBS (1 h, room temperature [RT]), membranes were exposed to a rat monoclonal anti-VEGF antibody (1:5,000, 512808; BioLegend, San Diego, CA, USA) at RT for 1 h followed by incubation at RT for 1 h with a horseradish peroxidase (HRP)-conjugated goat anti-rat immunoglobulin G (IgG)-peroxidase-conjugated antibody (1:5,000, 31470; Pierce Biotechnology, Waltham, MA, USA). Membranes were tested for monoclonal anti-PEDF (1:1,000; MAB1059; Millipore, Burlington, MA, USA) overnight at 4°C followed by incubation at RT for 2 h with a goat anti-mouse IgG-HRP (1:10,000; sc2005; Santa Cruz Biotechnology, Dallas, TX, USA). Signals were detected with an enhanced chemiluminescence (ECL) kit (ECL western blotting detection kit; GE Healthcare) and captured with ImageQuant 400 (GE Healthcare). The relative intensities of the immunoreactive bands were analyzed with ImageQuantTL software (GE Healthcare). The loading was verified by Ponceau S red (Sigma-Aldrich, St. Louis, MO, USA), and the same blot was stripped and reblotted with an anti-β-actin monoclonal antibody (Sigma-Aldrich) to normalize the VEGF and PEDF levels.
4
Immunostaining of Melanoma Cells
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Melanoma cells for immunostaining were kept in SlideFlasks (Thermo Fisher Scientific). Cells were fixed and immunostained as described previously for HEK293 cells.34 (link) Mouse anti-PEDF-antibody (MAB1059; Millipore, Hellerup, Denmark) was diluted 1:100, while the secondary antibody (568 Alexa Fluor Goat-anti-mouse, Thermo Fisher Scientific) was diluted 1:400 both in PBS with 0.5 w/v% BSA. Nuclei were stained with DAPI (Sigma-Aldrich).
5
Immunoblotting Procedure for Protein Analysis
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Cells were scraped into lysis buffer (150 μL/35-mm well) containing 20 mM HEPES (pH 7.4), 1% SDS, 150 mM NaCl, 1 mM EGTA, 5 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 100 μM sodium orthovanadate, 10 μg/mL leupeptin, and 10 μg/mL aprotinin. The lysate was resolved by SDS-PAGE, electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), and processed for immunoblot analysis. Antibodies used in this study were against PEDF (1:1000 dilution; MAB1059; Millipore), Bax, type I collagen 1A1, α-SMA, laminin-R, and Bcl-xL (1:1000 dilution; all from Santa Cruz Biotechnology), phospho-Stat3 (Tyr705), LRP6, PPARγ (1:1000 dilution; all from Cell Signaling Technology), PNPLA2/ATGL (1:1000 dilution; GTX59676; GeneTex, Inc) and cleaved caspase 3 (1:500 dilution; Abcam). Proteins of interest were detected using the appropriate IgG-HRP secondary antibody and ECL reagent. X-ray films were scanned on a Model GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA) and analyzed using Labworks 4.0 software. Blots from at least three independent experiments were used for quantification.
6
PEDF Expression in Transduced HUVECs
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HUVECs were transduced in six-well plate at 25% confluency with 20, 100, or 200 ng p24 LV/CMV-intron-PEDF-PE or 100 ng p24 LV/CMV-intron-AsRED-PE as negative control, and subsequently subcultured in T25 flasks. Harvest, lysis and western blotting were performed as described previously.40 (link) In short, cells were lysed 5 days after transduction in 100 µl of lysis buffer. 15 µg of total protein or 24 µl medium were electrophoresed on a 12% polyacrylamide gel (Mini-PROTEAN TGX gels; BioRad) together with the Precision Plus Protein Standard marker (BioRad). Following transfer to a Trans-Blot Turbo PVDF Membrane (Bio-Rad Laboratories), PEDF was detected using monoclonal mouse antipigment epithelium-derived factor antibody (MAB1059; Millipore). As a loading control, rabbit polyclonal antibody against β-actin (Abcam 8227–50; Abcam, Cambridge, MA) was used. Detection was performed using secondary horseradish peroxidase-conjugated polyclonal goat antimouse and polyclonal goat antirabbit antibodies, respectively (numbers P0447 and P0448; Dako, Glostrup, Denmark). Bound antibodies were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA) on an ImageQuant LAS4000 digital imaging system (GE Healthcare, Cleveland, OH).
7
Visualizing PEDF Expression in Transduced Cells
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HUVECs were transduced in six-well plates at 25% confluency with 100 ng p24 LV/CMV-intron-PEDF-PE or LV/CMV-intron-AsRED-PE and subsequently subcultured in slide flasks. Approximately 1 × 106 HEK-293 cells were transduced in p10 dishes with 5 ml LV/CMV-miR(5,B,7)-PEDF-PE. Five days after transduction, the cells were fixed in 4% paraformaldehyde (Hounisen, Risskov, Denmark) and stained with mouse monoclonal PEDF antibody (Millipore MAB1059) as described previously.39 (link) Detection was performed using secondary Alexa Flour 568 goat antimouse antibody (catalog number A11004; Invitrogen). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). Cells were visualized using a confocal laser scanning microscope (LSM 710, Zeiss, Jena, Germany).
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