Af1857

Manufactured by R&D Systems

Sourced in United States

AF1857 is a laboratory reagent manufactured by R&D Systems. It is a recombinant human protein that functions as a growth factor. The core function of AF1857 is to stimulate cellular processes, but a detailed description of its intended use is not available.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Nanodrop one, by Thermo Fisher Scientific (2 mentions) Ab155458, by Abcam (1 mentions) Ab14715, by Abcam (1 mentions) Chemidoc touch system, by Bio-Rad (1 mentions) Image lab software 5, by Bio-Rad (1 mentions) Complete ultra tablets, by Merck Group (1 mentions) Phosstop, by Merck Group (1 mentions) T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Gfap g a 5, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions) Bbs nc 6 h14, by Agilent Technologies (1 mentions) Bz x analysis software, by Keyence (1 mentions) Bz x710 microscope, by Keyence (1 mentions) A2228, by Merck Group (1 mentions) Sc 188, by Santa Cruz Biotechnology (1 mentions) C4606, by Merck Group (1 mentions) Sc 32233, by Santa Cruz Biotechnology (1 mentions) S2100, by Solarbio (1 mentions) Haf109, by R&D Systems (1 mentions)

20 protocols using af1857

Quantification of Lung Lipocalin-2 and MPO

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For optimal protein extraction, total left lung homogenate (prepared as described above) was added to 5 × RIPA buffer with protease inhibitors, incubated on ice for 30 min. and centrifuged. Supernatant was collected and ELISA was performed. Quantification of plasma and lung lipocalin-2 was executed by standard ELISA techniques (10 (link), 24 (link)). The following antibodies was used: Affinity-purified goat anti-lipocalin-2 (1:500, AF 1857, R&D Systems), biotinylated rabbit anti-lipocalin-2 [1:200, in-house-generated antibody (10 (link))], and horseradish peroxidase Avidin D (1:3,000, 18–4,100, eBioscience). As a surrogate marker of neutrophil influx myeloperoxidase, (MPO) ELISA (ab155458, Abcam) was performed according to manufacturer's instructions.

Dahl S.L., Woodworth J.S., Lerche C.J., Cramer E.P., Nielsen P.R., Moser C., Thomsen A.R., Borregaard N, & Cowland J.B. (2018). Lipocalin-2 Functions as Inhibitor of Innate Resistance to Mycobacterium tuberculosis. Frontiers in Immunology, 9, 2717.

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Western Blot Analysis of Intestinal Proteins

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Proteins were extracted from mouse ileum by homogenizing in T-PER Tissue Protein Extraction Reagent (Thermo Fischer, 78510) supplemented with a protease inhibitor cocktail (cOmplete ULTRA Tablets, Sigma-Aldrich; 5892953001) and a phosphatase inhibitor (PhosSTOP, Sigma-Aldrich; 4906845001). 40 μg of total protein was loaded onto 4–20% gradient SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20. For detection with anti-STAT3 antibodies, membranes were blocked with 5% BSA in PBS with 0.1% Tween-20. Membranes were incubated at room temperature for one hour with the following primary antibodies: anti-REG3G antiserum raised against recombinant REG3G (Cash et al., 2006 (link)) and anti-succinate dehydrogenase (Abcam; ab14715), and at 4°C overnight with the following antibodies: anti-STAT3 (Cell Signaling; 4904S), anti-phospho-STAT3 (Tyr705) (Abcam; ab76315), and anti-lipocalin-2 (R&D systems; AF1857). After washing with PBS with 0.1% Tween, membranes were incubated with HRP-conjugated secondary antibodies. Membranes were visualized using a Bio-Rad ChemiDoc^™ Touch system, and band density was quantified using Bio-Rad Image Lab Software 5.2.1.

Brooks JF I.I., Behrendt C.L., Ruhn K.A., Lee S., Raj P., Takahashi J.S, & Hooper L.V. (2021). The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock. Cell, 184(16), 4154-4167.e12.

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Immunohistochemical Analysis of Retinal Proteins

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Polyclonal goat anti-Lcn2 (AF1857; R&D systems), monoclonal mouse anti-neuron specific enolase (NSE; BBS/NC/VI-H14; Dako, Glostrup, Denmark), monoclonal mouse anti-glial fibrillary acidic protein (GFAP; G-A-5; Sigma-Aldrich) and monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (G3pdh; Medical and Biological Laboratories, Aichi, Japan) antibodies were the primary antibodies used in this study. Peroxidase-conjugated antibodies (GE Healthcare, IL, United States) or fluorescent-conjugated antibodies (Jackson ImmunoResearch, PA, United States) were used as secondary antibodies for immunoblotting or immunohistochemistry, respectively. Immunoblotting, TdT-mediated dUTP nick and labeling (TUNEL) assay, and immunohistochemical analysis of Lcn2 were carried out as described previously (Ueno et al., 2018 (link)). For the immunohistochemical analysis of GFAP, paraformaldehyde-fixed retinae were immersed in increasing concentrations of sucrose (10–30%) and embedded in frozen medium; cryosections (5 μm thickness) were further treated with 0.1% Triton X-100. TUNEL positivity, GFAP immunoreactivity, and nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Dojindo Laboratories, Kumamoto, Japan) were visualized with a BZ-X710 microscope (Keyence, Osaka, Japan) and measured using the BZ-X analysis software (Keyence).

Yoneshige A., Hagiyama M., Takashima Y., Ueno S., Inoue T., Kimura R., Koriyama Y, & Ito A. (2021). Elevated Hydrostatic Pressure Causes Retinal Degeneration Through Upregulating Lipocalin-2. Frontiers in Cell and Developmental Biology, 9, 664327.

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Antibody Characterization and Validation

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The following antibodies were used for immunohistochemical, immunofluorescence and Western blot analyses: sheep polyclonal antibody against uromodulin (ab9029, Abcam, Cambridge, UK) (1:200 for IF and for IHC); rat monoclonal antibody against HA (#11 867 423 001, Roche) (1:500 for IF and 1:1,000 for WB); rabbit polyclonal antibody against calreticulin (C4606, Sigma-Aldrich) (1:500 for IF); rat antibody against mouse F4/80 (MCA497GA, Serotec, BioRad)(1:50 for IF and IHC); rabbit polyclonal antibody against Atf3 (sc-188, Santa Cruz Biotechnology, Dallas, TX) (1:50 for IF and 1:500 for WB); goat polyclonal against Lipocalin 2/NGAL (AF1857, R & D Systems, Bio-Techne, Minneapolis, MN) (1:500 for WB); rabbit polyclonal against Kim1 (TIM-1) (NBP1-76701, Novus Biologicals, Bio-Techne) (1:500 for WB); mouse monoclonal antibody against β-Actin (A2228, Sigma-Aldrich) (1:20,000 for WB); mouse monoclonal antibody against Gapdh (sc-32233, Santa Cruz Biotechnology) (1:20,000 for WB).

Trudu M., Schaeffer C., Riba M., Ikehata M., Brambilla P., Messa P., Martinelli-Boneschi F., Rastaldi M.P, & Rampoldi L. (2017). Early involvement of cellular stress and inflammatory signals in the pathogenesis of tubulointerstitial kidney disease due to UMOD mutations. Scientific Reports, 7, 7383.

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Immunohistochemical Analysis of Brain Tissue

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Mice were killed and perfused with saline then followed by 4% paraformaldehyde. Brains were removed and post fixed at 4 °C overnight. Brains were dehydrated in sucrose solution (30%) for 24 h at least. Coronal sections (35 μm thick) were cut on a cryostat and stored in cryoprotectant at −20 ºC. In brief, sections were washed in PBS buffer, followed by incubation with blocking medium (PBS buffer containing 10% donkey serum, 1% bovine albumin and 0.1% Triton X-100) for 1 h at room temperature. Then the sections were incubated with primary antibodies (IbaI, 1:500, Novus, #NBP2-19019; GFAP, 1:400, CST, #12389; NeuN, 1:400, CST, #24307; LCN2, 1:500, R&D Systems, #AF1857) in PBS containing 1% BSA at 4 ºC overnight. After washing 4 times for 5 min in PBS, sections were incubated with secondary antibody (TRITC-conjugated donkey anti-rabbit or AF488-conjugated donkey anti-goat, 1:400) for 1 h at room temperature. Sections were then washed in PBS, incubated with DAPI (1:1000, CST, #4083) in PBS for 10 min. Subsequently, sections were washed in PBS buffer, mounted onto microscope slides with anti-fade mounting medium (Solarbio, #S2100).

Xiang X., Tang X., Yu Y., Xie S., Liu L., Chen M., Zhang R., Kang X., Zheng Y., Yang G., Gan S, & Zhu S. (2022). Role of lipocalin-2 in surgery-induced cognitive decline in mice: a signal from neuron to microglia. Journal of Neuroinflammation, 19, 92.

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Quantification of Serum and Fecal Biomarkers

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LCN2, IL6, and IL22 in serum and feces were quantified with enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s instructions. Measurements were performed using Mouse Lipocalin2 DuoSet (DY1857-05; R&D, Minneapolis, MN), Mouse IL6 DuoSet (DY406-05; R&D), and the IL22 Mouse ELISA Kit (88-7422-22; Invitrogen). LCN2 expression in both the intestine and liver were measured by Western blot analysis. The primary antibody, goat anti-LCN2 (1:500, AF1857; R&D), was incubated with a polyvinylidene difluoride membrane at 4°C overnight. The secondary antibody, anti-goat IgG Heavy and light chains (H+L) (1:3000, HAF109; R&D), was incubated with a polyvinylidene difluoride membrane for 1 hour at room temperature.

Zhang A., Sodhi C.P., Wang M., Shores D.R., Fulton W., Prindle T., Brosten S., O’Hare E., Lau A., Ding H., Jia H., Lu P., White J.R., Hui J., Sears C.L., Hackam D.J, & Alaish S.M. (2020). A Central Role for Lipocalin-2 in the Adaptation to Short-Bowel Syndrome Through Down-Regulation of IL22 in Mice. Cellular and Molecular Gastroenterology and Hepatology, 10(2), 309-326.

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Comprehensive Antibody Panel for Oxidative Stress Analysis

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The following primary antibodies were used for immunoblot analysis and immunohistochemistry: mouse monoclonal anti–transferrin receptor antibody (13‐6800, Invitrogen), goat polyclonal anti–Lipocalin‐2/NGAL antibody (AF1857, R&D systems), rabbit monoclonal anti–β‐actin (ACTB) antibody (AC026, ABclonal), rabbit monoclonal anti–IRP1 antibody (ab126595, Abcam), rabbit polyclonal anti–IRP2 antibody (NB100‐1798, Novus), rabbit polyclonal anti–FTH1 antibody (3998, CST), rabbit polyclonal anti–ferritin light chain (FTL) antibody (ab69090, Abcam), rabbit polyclonal anti–divalent metal transporter 1 (DMT1) antibody (20507‐1‐AP, proteintech), rabbit polyclonal anti–8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) antibody (bs1278R, Bioss), rabbit polyclonal anti–4‐Hydroxynonenal antibody (bs6313R, Bioss), anti–CD10 antibody (ab256494, Abcam), anti–Ki67 antibody (ab16667, Abcam), rabbit monoclonal anti–Cleaved Caspase‐3 antibody (9664, CST), rabbit monoclonal anti–Glutathione Peroxidase 4 antibody (ab125066, Abcam), and anti–xCT antibody (NB300‐318, Novus). Secondary antibodies used in immunoblot analysis and immunohistochemistry were as follows: HRP‐conjugated polyclonal Goat anti–Rabbit antibody (P0448, DAKO), HRP‐conjugated polyclonal Rabbit anti–mouse antibody (P0260, DAKO) and HRP‐conjugated polyclonal Rabbit anti–Goat antibody (P0160, DAKO).

Cheng Z., Akatsuka S., Li G.H., Mori K., Takahashi T, & Toyokuni S. (2021). Ferroptosis resistance determines high susceptibility of murine A/J strain to iron‐induced renal carcinogenesis. Cancer Science, 113(1), 65-78.

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Liver Protein Expression Analysis

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Western blot assays were performed using whole liver lysates13 or nuclear extracts of the liver samples,14, 15 as described previously. LCN2 antibodies were purchased from R&D Systems (AF1757 and AF1857; Minneapolis, MN). SREBP‐1 antibody was purchased from Santa Cruz Biotechnology (SC‐8984; Dallas, TX). Acetyl‐CoA carboxylase (ACC) antibody (3662S) and caspase 3 (CASP3) antibodies (cleaved and total (9661 or 9662) were purchased from Cell Signaling Technology (Danvers, MA). Carboxylesterase 1 (CES1; ab45957), carboxylesterase 2 (CES2; ab56528), and Tubulin (ab4074) antibodies were purchased from Abcam (Cambridge, United Kingdom).

Xu Y., Zhu Y., Jadhav K., Li Y., Sun H., Yin L., Kasumov T., Chen X, & Zhang Y. (2019). Lipocalin‐2 Protects Against Diet‐Induced Nonalcoholic Fatty Liver Disease by Targeting Hepatocytes. Hepatology Communications, 3(6), 763-775.

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Kidney Histological Analysis Protocol

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Following euthanasia, kidneys were removed and incubated at room temperature overnight in fixative containing 3.7% formaldehyde, 10 mM sodium m-periodate, 40 mM phosphate buffer, and 1% acetic acid. The fixed kidney was dehydrated through a graded series of ethanols, embedded in paraffin, sectioned (5 μm), and mounted on glass slides. Immunostaining was carried out as in previous reports (7 (link)). Antibodies used include: anti-KIM1 (AF1817, R&D Systems), anti-NGAL (AF1857, R&D Systems), anti-CD68 (ab125212, Abcam), anti-αSMA (A5228, MilliporeSigma), anti–type 1 collagen (600-401-103-0.1, Rockland), and anti–type 4 collagen (600-401-106-0.1, Rockland). For calculation of percentage area, 5 fields on the kidney section were randomly selected, and the ratios of DAB-positive area to total areas in each field were counted in each power field using ImageJ software (NIH). For CD68 and CD3 staining, the number of positive cells was counted in a blinded fashion. Quantification was performed using ImageJ software as previously reported (27 (link)).

Sasaki K., Terker A.S., Tang J., Cao S., Arroyo J.P., Niu A., Wang S., Fan X., Zhang Y., Bennett S.R., Zhang M.Z, & Harris R.C. (2022). Macrophage interferon regulatory factor 4 deletion ameliorates aristolochic acid nephropathy via reduced migration and increased apoptosis. JCI Insight, 7(4), e150723.

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Comprehensive Antibody Validation Protocol

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Antibodies against Gbp2 (ab203238), GFAP (ab53554, ab7260), LCN2 (ab63929), LRP1 (ab92544), MAG (ab89780), MBP (ab40390), NF200 (ab7795), PLP (ab28486) and S100A10 (ab76472) were purchased from Abcam, UK; antibody against CD31 (550274) was purchased from BD Biosciences, USA; antibodies against GFAP (3670S), GFP (2955S, 2956S), p38 (9212S), pp38 (4511S) and β-actin (8457S) were purchased from Cell Signaling Technology, USA; antibodies against CC1 (OP80), dMBP (AB5864) and Olig2 (AB9610) were purchased from Millipore, USA; antibodies against C3d (AF2655), Galectin3 (AF1197), LAMP1 (AF4320) and LCN2 (AF1857) were purchased from R&D Systems, USA; antibodies against Gbp2 (sc-166960) and LRP1 (sc-57351) were purchased from Santa Cruz Biotechnology, USA; antibody against pLRP1 (PA5-101013) was purchased from Thermo Fisher, USA; antibody against Iba1 (019-19741) was purchased from Wako, Japan. All antibodies were used at a dilution of 1:50 to 1:2000 for immunofluorescence and 1:800 to 1:5000 for immunoblotting according to the manufacturer’s instructions. Alexa Fluor 488, 594 and 647 conjugated secondary antibodies were purchased from Jackson, USA.

Wan T., Zhu W., Zhao Y., Zhang X., Ye R., Zuo M., Xu P., Huang Z., Zhang C., Xie Y, & Liu X. (2022). Astrocytic phagocytosis contributes to demyelination after focal cortical ischemia in mice. Nature Communications, 13, 1134.

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