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Anti rabbit and anti mouse secondary antibodies

Manufactured by Jackson ImmunoResearch
Sourced in United States, Cameroon

Jackson ImmunoResearch's anti-rabbit and anti-mouse secondary antibodies are targeting and binding to primary antibodies raised in rabbit or mouse, respectively. These secondary antibodies are designed to facilitate detection and visualization of the target antigen in various immunoassays and microscopy applications.

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13 protocols using anti rabbit and anti mouse secondary antibodies

1

Prostate Cancer Cell Line Maintenance and Treatment

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The prostate cancer cell lines, LNCaP and PC-3, were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium containing 10% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO2. For experiments, LNCaP cells were plated on poly-l-lysine-coated culture flasks at a density of 12000 cells per cm2 surface area for 24 h, followed by treatment with test agents in serum-free RPMI 1640 medium. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from TCI America (Portland, OR) and the ECL western blotting system from GE Healthcare Life Sciences (Pittsburgh, PA). Antibodies specific for the following protein targets were used: phospho-S473-Akt, phospho-T308-Akt, Akt, PDK-1, and β-actin (Cell Signaling Technology, Inc., Beverly, MA), PHLPP (Novus Biologicals, Littleton, CO), and Na+-K+ ATPase and ILK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Alexa Fluor 555- and 488-conjugated goat antirabbit and antimouse IgG were purchased from Invitrogen (Carlsbad, CA), and antimouse and antirabbit secondary antibodies was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
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2

Grape Seed Proanthocyanidin Protects Pancreatic Cells

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Grape seed proanthocyanidin (purity exceeds 96%, Lot no. G050412) was purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China). STZ was purchased from Sigma. TUNEL staining kit (in situ Cell Death Detection kit) was obtained from Roche Diagnostics, (Indianapolis, IN, USA). Primary antibodies used in this study include: Rabbit anti-Caspase-12, rabbit anti-GRP78 (Abcam Ltd., Hong Kong, China), rabbit anti-p-ERK, rabbit anti-ERK antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
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3

Antibody Validation for β-Catenin Signaling

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The antibodies that were used include: mouse anti-β-catenin (IB: 1:5000; IF: 1:300; BD Transduction Laboratories, Franklin Lakes, NJ, USA), mouse anti-active β-catenin (IB: 1:1000; IF: 1:150; Anti-ABC clone 8E7; Merck Millipore, Temecula, CA, USA), rabbit anti-GFP (1:1000; Santa Cruz Biotechnology), rat anti-HA (IB: 1:2500; IF: 1:300; Roche, Indianapolis, IN, USA), mouse anti-FLAG (1:5000; Sigma), mouse anti-HTRA1 (IB: 1:500; IF: 1:50; IHC 1:50; R&D Systems, Minneapolis, MN, USA), rabbit anti-HTRA1 (IF: 1:50; IB 1:500; PRSS11 (C-term), Acris Antibodies GmbH), rabbit anti-HTRA1 (IF: 1:50; IB 1:1000; Abgent, Inc.) rabbit anti-Golgin-97 (IF: 1:100; (D8P2K) Cell Signaling Technology, Inc.). Mouse anti-tubulin (1:10000; Sigma) was used as a loading control. Anti-rat horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (1:5000). Anti-mouse and anti-rabbit secondary antibodies were obtained from Jackson Immuno Research (West Grove, PA, USA) (1:10000). For IF, Alexa red and green (1:500; Molecular Probes, Grand Island, NY, USA) were used.
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4

Breast and Prostate Cancer Cell Lines

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The MDA-MB-231 and MDA-MB-468 breast cancer and PC-3 prostate cancer cell lines were purchased from American Type Culture Collection (Manassas, VA). Media used for the maintenance of these cells were as follows: MDA-MB-231 and MDA-MB-468, DMEM; PC-3, RPMI 1640, all of which were supplemented with 10% fetal bovine serum. Cells were incubated at 37°C in a humidified incubator containing 5% CO2. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] was obtained from TCI America (Portland, OR), and the ECL Western Blotting System from GE Healthcare Life Sciences (Pittsburgh, PA). Antibodies specific for the following protein targets were used: p-Thr172-AMPK, AMPK, p-Thr389-p70S6K, p70S6K, Ser473-Akt, Akt, Foxo3a, claudin-1, vimentin, snail, and β-actin (Cell Signaling Technology, Inc., Beverly, MA); E-cadherin, BD Biosciences (San Diego, CA). Alexa Fluor 555- and 488-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Invitrogen (Carlsbad, CA), and anti-mouse and anti-rabbit secondary antibodies was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
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5

Grape Seed Extract Induces ER Stress-Mediated Apoptosis

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CP was purchased from Sigma-Aldrich (St. Louis, MO, USA). Grape seed proanthocyanidin extract (purity > 96%, lot no. G050412) was purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China). Primary antibodies used in this study were: rabbit anti-GRP78, rabbit anti-caspase-12 (Abcam Ltd., Hong Kong, China), rabbit anti-p-ERK, rabbit anti-ERK (Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse anti-β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). The In Situ Cell Death Detection kit (Roche Diagnostics, Indianapolis, IN, USA) was used for the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.
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6

Serum Amyloid A1 Quantification Protocol

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Human Serum Amyloid A1 DuoSet ELISA was from R&D systems (DY3019-05). Recombinant Human Serum Amyloid A1 was from Novus Biologicals (NBP2-34875). Recombinant Human TRAP6 was from Tocris (cat #3497). Fibrinogen from human plasma (cat #F3879) and anti-Tubulin antibody (1:10,000), prostaglandin E1 (P5515), p-Nitrophenyl Phosphate (487663) were all purchased from Sigma-Aldrich Israel. Anti-Serum Amyloid A antibody was from Abcam (1:3000; cat # ab687), Anti-Integrin αIIbβ3 was from Santa Cruz (sc-21783), and Anti-CD41 was from ThermoFisher (cat # 11-0411-82). Anti-mouse and anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch (1:10,000). The RGD-containing peptide GRGDSP and scrambled control peptide GRGESP were synthesized at the Blavantik center for drug discovery at Tel Aviv University.
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7

Investigating PAR-2 Signaling Pathway

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Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) and 0.25% Trypsin–EDTA solution were purchased from Gibco-BRL (Grand Island, NY, USA). Tryptase was purchased from Sigma-Aldrich (St. Louis, MO, USA), and it is the human lung Tryptase, which is a neutral serine protease and the predominant protein in mast cell granules. PAR-2 inhibitor FSLLRY-NH2 (FS) was synthesised by CL Bio-Scientific Inc. (Xi An, China). CCK-8, RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). Rabbit anti-PAR-2 polyclonal antibody and fluoroshield mounting medium with 4′,6-diami-dino-2-phenylindole (DAPI) were purchased from Abcam (Hongkong, China). Anti-TLR4 monoclonal antibody, anti-VCAM-1 antibody (EPR5 047) and anti-occludin antibody (EPR8208) were purchased from Abcam (Hongkong, China). Anti-GAPDH antibody was purchased from Bioworld Technology, Inc. (USA). Anti-p44/42 MAPK monoclonal antibody (extracellular regulated protein kinases, ERK), anti-Phospho-p44/42 monoclonal antibody (phosphoERK) and NF-kappa B were purchased from Cell Signaling (Beverly, MA, USA).Anti-rabbit and anti-mouse secondary antibodies were all purchased from Jackson Immuno Research Laboratories Inc. (Boston, MA, USA).
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8

Immunohistochemical Profiling of Immune Cells

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Immunohistochemistry was performed on 5 μm-thick FFPE tissue sections as previously indicated1 (link),39 (link). Tissues were stained with combinations of primary antibodies including polyclonal rabbit anti-IFNγ (Abcam, Cambridge, MA), rabbit anti-TNF (Bioss Antibodies, Boston, MA), rabbit anti-IL-4 (Abcam), rabbit anti-IL-10 (Abcam), rabbit anti-CD3 (Agilent, Santa Clara, CA), mouse anti-calprotectin (clone: MAC378, ThermoFisher), mouse anti-CD163 (clone: 1D6, ThermoFisher), and mouse anti-human alveolar macrophage antibody (HAM56, Enzo Life Sciences, Farmingdale, NY). Tissues were subsequently stained with appropriate anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 hr at room temp, and coverslips were mounted with DAPI-containing Prolong Gold mounting medium (ThermoFisher). Cells were imaged on an Olympus confocal microscope (Olympus, Waltham, MA) running FlowView 1000 software maintained by the University of Pittsburgh’s Department of Microbiology and Molecular Genetics, or a Nikon e1000 epifluorescence microscope running Nikon Elements (Nikon Instruments, Melville, NY). Images were annotated with Photoshop CS5.1 (Adobe Systems, San Jose, CA) and projections of z-stacks were made with the FIJI build of ImageJ42 (link). Quantitative image analysis was performed with CellProfiler43 (link).
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9

Western Blot Analysis of Key Molecular Markers

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For cell lysis, the harvested samples were incubated on ice in whole cell extract lysis buffer for 30 minutes. Lysates were centrifuged at 12,000 rpm for 10 minutes, and the protein concentration was measured using the Bradford assay (Bio‐Rad, Hercules, CA, USA). For western blotting, 15–100 μg of lysates (depend on the target proteins assayed) were then boiled for 5 minutes with sample buffer before being separated on SDS‐polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk/TBST buffer. The primary antibodies used were as follows: HER2, beta‐actin (Merck KGaA), ERα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), progesterone receptor (PR) A/B, epidermal growth factor receptor (EGFR), and PTEN (Cell Signaling Technology, Beverly, MA, USA). Anti‐rabbit and anti‐mouse secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).
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10

Immunohistochemical Analysis of Abdominal Aortic Aneurysm

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After directly placing in saline upon excision of the AAA during surgery, human specimens were fixed in 10% formalin, embedded in OCT compound, frozen, and sectioned. Frozen sections were hydrated in PBS, and blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Abcam, 1:400 and anti-CD68, Millipore Sigma, 1:100) or control IgG (anti-mouse and anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in PBS, mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) and Verhoeff-Van Gieson (VVG) stains were performed on serial sections to analyze morphology and severity of the AAA tissues.
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