Cells, seeded overnight on cover-slides, were incubated with increasing concentrations of inhibitors, and stimulated with TNFα (2 ng/ml) for 30 min. Cells were fixated with 2% paraformaldehyde, washed, and permeabilized with PBS/Tween®20. Cells were stained using primary antibodies for NF-κB (p65), (rabbit pAb, Santa Cruz, Cat. No.: sc-372, 1:1,000) and fluorescence-labeled secondary antibody [Alexa Fluor® 488 goat anti-rabbit IgG (H+L), Life Technologies Cat. No.: 11008, 1:2,000] for 30 min. Subsequently, the cell nuclei were stained using DAPI (1:40,000, Life Technologies, Cat. No.: D1306) for 30 min. The cover-slides were embedded with Flouramount G (Invitrogen), dried overnight at 4°C, and evaluated by confocal microscope (Leica, LSM3).
Rabbit pab
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit pAb is a polyclonal antibody produced in rabbits. Polyclonal antibodies are a mixture of immunoglobulin molecules that recognize different epitopes of the target antigen. The core function of Rabbit pAb is to serve as a research tool for the detection and analysis of target proteins in various applications, such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 labeled anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg h l alexa fluor 594 labeled antibody, by Santa Cruz Biotechnology (2 mentions)
Zeis lsm 5 exciter confocal laser scanning microscope, by Zeiss (2 mentions)
Foxm1, by Santa Cruz Biotechnology (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Anti β catenin, by BD (2 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Mouse anti v5 mab, by Thermo Fisher Scientific (1 mentions)
Species specific secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Rabbit pab, by Cell Signaling Technology (1 mentions)
Rabbit pab, by Merck Group (1 mentions)
E9884, by Merck Group (1 mentions)
Cw0130, by CWBIO (1 mentions)
Rabbit pab, by Proteintech (1 mentions)
5 protocols using rabbit pab
1
Quantifying NF-κB Activation in Cells
2
Immunofluorescence Assay for β-Catenin and FOXM1
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Cells were grown on poly-L-Lysine-treated coverslips and after 6h of the different treatments the culture media was discarded, cells were washed thrice in PBS and permeabilized in methanol. After washing with PBS, coverslips were incubated with Anti-b-Catenin (1/250) mouse mAb (BD) and FOXM1 (1/250) rabbit pAb (Santa Cruz Biotechnology), for 1h at room temperature. Coverslips where washed thrice in PBS and incubated for 1h at room temperature with an anti-mouse IgG alexa fluor 488-labeled antibody (1/500) (Molecular Probes, Eugene, OR, USA) and with anti-rabbit IgG (H+L) alexa fluor 594-labeled antibody (1/500) (Santa Cruz Biotechnology). Finally, cells were incubated with 300 nM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS for 5 minutes at room temperature. Coverslips were mounted using Aqua Poly/Mount (Polysciences, Warrington, PA, USA) and visualized in a Zeis LSM 5 Exciter Confocal laser-scanning microscope (Zeiss, Germany). Images were analyzed using the image-J software (National Institutes of Health).
3
Immunofluorescent Labeling of Protein Markers
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DUX4-FL was detected with rabbit anti-DUX4-FL mAb E5524 (link) used at 1:200 dilution (Epitomics, Burlingame, CA). Myosin heavy chain (MyHC) isoforms were detected with mouse mAbs F59 or MF20 (Developmental Studies Hybridoma Bank, Iowa City, IA) used at 1:10 dilution of hybridoma supernatant. Activated caspase-3 was detected with a rabbit pAb (Cell Signaling Technologies, Beverly, MA) used at 1:100. TDP-43 was detected with either rabbit anti-TARDBP pAb (cat. 10782-2-AP; Proteintech, Chicago, IL) or mouse anti-TDP-43 mAb (cat. 60019-2; Proteintech). Ubiquitinated proteins were detected with mouse mAb FK2 (MBL, Woburn, MA) which reacts with K29-, K48-, and K63 mono- and poly-ubiquitinated proteins, but not free ubiquitin. V5 epitope tag was detected using either mouse anti-V5 mAb (Life Technologies, Grand Island, NY) or a rabbit pAb (EMD Millipore). GFP was detected with a rabbit pAb (Santa Cruz Biotechnology, Dallas, TX). Primary antibody binding was visualized with appropriate species-specific secondary antibodies (Life Technologies) conjugated to either Alexa Fluor 488 or Alexa Fluor 594 and used at 1:500. Nuclei were stained with bisbenzimide.
4
Immunohistochemical Analysis of miR-129-5p Inhibitor Effects on Osteogenic Genes in Mouse Calvaria
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To investigate the rescue effect of miR-129-5p inhibitor on osteogenic gene and TCF4 levels in mice calvarias, immunohistochemical staining analysis was performed as previously described (Yin et al., 2019 (link)). Mouse calvarias were dissected and fixed in 4% paraformaldehyde, decalcified in 17% ethylenediaminetetraacetic acid (Sigma, E9884) for 21 days, and embedded in paraffin (Huayong, Shanghai, China). Sections (5 μm in thickness) were dewaxed, immersed in the distilled water, blocked in 5% goat serum (CWBIO, CW0130) in PBS, and then incubated overnight at 4°C with primary antibodies against OCN (Rabbit pAb, 1:200, Santa Cruz Biotech, sc-365797, Dallas, TX, United States), OSTERIX (Rabbit pAb, 1:50; Proteintech, 12593-1-AP), and TCF4 (Rabbit pAb, 1:100, Proteintech, 22337-1-AP), respectively. Following three washes in PBS, the sections were labeled with HRP-labeled secondary antibody 1.5 h at room temperature and developed for color reaction using diaminobenzidine (CWBIO, CW2068) and hematoxylin counterstain. Slides were scanned by Aperio AT2 Digital Pathology Scanner (Leica), and protein immunostaining intensities on top surface of the calvarias were analyzed by Image-Pro Plus 6.0 software.
5
Visualization of β-Catenin and FOXM1 Expression
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Cells were grown on poly-L-Lysine-treated coverslips to form a nearly confluent monolayer and after 6h of the different treatments. Then, the culture media was discarded, cells were washed thrice in PBS and permeabilized in methanol. After washing with PBS, coverslips were incubated with Anti-β-Catenin (1/250) mouse mAb (BD) and FOXM1 (1/250) rabbit pAb (Santa Cruz Biotechnology), for 1h at room temperature, washed thrice in PBS and labeled with an anti-mouse IgG alexa fluor 488-labeled antibody (1/500) (Molecular Probes, Eugene, OR, USA) and with anti-rabbit IgG (H+L) alexa fluor 594-labeled antibody (1/500) (Santa Cruz Biotechnology), for 1h at room temperature. Finally, cells were incubated with 300 nM 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS for 5 minutes at room temperature. Coverslip were mounted using Aqua Poly/Mount (Polysciences, Warrington, PA, USA) and visualized in a Zeis LSM 5 Exciter Confocal laser-scanning microscope (Zeiss, Germany). Images were analyzed using the image-J software (National Institutes of Health).
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