After various treatments, the cells were washed with PBS and lysed with RIPA lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02 % sodium azide, 0.1 % sodium dodecyl sulfate (SDS), 1 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Equal amounts of protein from the different treatment groups were separated by SDS-polyacrylamide gel electrophoresis (10 % gel) and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were blocked with 4 % skim milk in Tris-buffered saline Tween-20 (TBST) and then incubated with primary antibodies (goat anti-PTP1B (1:500 dilution, Santa Cruz Biotechnology), polyclonal rabbit anti-phospho- or total forms of Src (1:1000 dilution, Cell signaling), rabbit anti-IκB (1:1000 dilution, Santa Cruz), mouse anti-β-actin (1:2000 dilution, Sigma-Aldrich), mouse anti-α-tubulin (1:5000 dilution, Sigma-Aldrich), and goat anti-LCN2 (1:500 dilution, R&D systems)). After thorough washing with TBST, horseradish peroxidase-conjugated secondary antibodies were applied. The blots were developed using an enhanced chemiluminescence detection kit (SuperSignal™ West Femto, ThermoFisher).
Goat anti lcn2
Manufactured by R&D Systems
Sourced in Japan, United States
Goat anti-LCN2 is an antibody product designed for use in various research applications. It targets the LCN2 (Lipocalin-2) protein, which is involved in a range of biological processes. This antibody can be utilized in techniques such as western blotting, immunohistochemistry, and ELISA to detect and study the LCN2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Merck Group (2 mentions)
Mouse anti gfap, by Santa Cruz Biotechnology (2 mentions)
Mouse anti stat3, by Cell Signaling Technology (2 mentions)
Py705 stat3, by Cell Signaling Technology (2 mentions)
Axioskop microscope, by Zeiss (2 mentions)
Rabbit anti tcf4, by Cell Signaling Technology (2 mentions)
Rabbit anti tcf1, by Cell Signaling Technology (2 mentions)
Mouse anti β catenin, by Merck Group (2 mentions)
Rat anti cd104, by BD (2 mentions)
Fitc or rrx conjugated secondary abs, by Jackson ImmunoResearch (2 mentions)
Rabbit anti lef1, by Cell Signaling Technology (2 mentions)
Immpact dab, by Vector Laboratories (2 mentions)
M o m kit, by Vector Laboratories (2 mentions)
Mouse anti myc tag, by Zymo Research (2 mentions)
Rabbit anti iκb, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Rabbit anti erk2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti glur1, by Merck Group (1 mentions)
9 protocols using goat anti lcn2
1
Western Blot Analysis of Protein Targets
2
Detailed Immunoblotting and Immunofluorescence Antibodies
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The following antibodies were used for immunoblotting: Mouse anti-calbindin (Sigma), rabbit anti-EAAT2 (Cell Signaling Technology), mouse anti-NeuN (Millipore), Rabbit anti-GluR1 (Millipore), rabbit anti-EAAT4 (Abcam) and goat anti-Lcn2 (R&D Systems). Goat anti-IKK2 (human specific, detects only the transgene), rabbit anti-IKK1/2, rabbit anti-ERK2, rabbit anti-EAAT1, rabbit anti-EAAT3, rabbit anti-galectin 3 (Mac-2), mouse anti-GFAP and all corresponding HRP coupled secondary antibodies were obtained from Santa Cruz Biotechnologies.
Rabbit anti-Prosap1 was kindly provided by Prof. Tobias Böckers (Ulm, Germany).
Goat anti-IKK2, mouse anti-GFAP, mouse anti-NeuN and Mouse anti-calbindin were also used for immunofluorescence. Additionally, following antibodies were used for immunofluorescence: mouse anti-Aldh1l1 (Abcam), rabbit anti-RelA (Santa Cruz Biotechnologies), PE-labeled rat anti-CD11b (eBioscience), and rat anti-CD45, rat anti-CD8, rat anti CD4 (all from BD Biosciences), rabbit anti-Iba1 (Wako) and chicken anti-GFP (Abcam). Corresponding Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Life Technologies).
Rabbit anti-Prosap1 was kindly provided by Prof. Tobias Böckers (Ulm, Germany).
Goat anti-IKK2, mouse anti-GFAP, mouse anti-NeuN and Mouse anti-calbindin were also used for immunofluorescence. Additionally, following antibodies were used for immunofluorescence: mouse anti-Aldh1l1 (Abcam), rabbit anti-RelA (Santa Cruz Biotechnologies), PE-labeled rat anti-CD11b (eBioscience), and rat anti-CD45, rat anti-CD8, rat anti CD4 (all from BD Biosciences), rabbit anti-Iba1 (Wako) and chicken anti-GFP (Abcam). Corresponding Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Life Technologies).
3
Immunohistochemical and Immunofluorescence Analysis
4
Triple Immunostaining of Brain Tissue
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Triple immunostaining was performed as follows. Free-floating brain tissue sections were incubated simultaneously with mouse anti-GFAP (1:500; Santa Cruz Biotechnology), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1, 1:150; Wako, Osaka, Japan), and goat anti-LCN2 (1:200; R&D Systems). To distinguish between the mouse primary antibody and endogenous mouse immunoglobulins in the mouse brain, we used the Mouse on Mouse (MOM) immunodetection kit (Vector Laboratories, CA, USA). Samples were then washed three times with 0.1 M PBS and incubated with Alexa Fluor 488-, 594-, and 790-conjugated donkey anti-mouse, anti-rabbit, or anti-goat secondary antibody (Invitrogen Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Fluorescence was visualized with a BX51-DSU microscope (Olympus) and digital images were captured.
5
Visualizing Neutrophil and Macrophage LCN2 Expression in Liver
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To examine the expression and distribution of LCN2, 5-µm cryostat liver sections were incubated with one or two primary antibodies overnight: goat anti-LCN2 (1:100; R&D) and rat anti-Ly6G/6C (a marker of neutrophils; 1:100; BD Diagnostic Systems) or goat anti-LCN2 and rat anti-F4/80 (a marker of macrophages; 1:100; Bio-Rad Laboratories Inc.). The secondary antibodies were Alexa Fluor 488-conjugated donkey anti goat or rat IgG (1:200) or Alexa Fluor 555-conjugated donkey anti-goat or rat IgG (1:200) from the Jackson ImmunoResearch Laboratories (West Grove, PA, USA). As a negative control, the sections were exposed to nonimmune IgG (in replacement of primary antibodies) with the same secondary antibodies, and no specific staining was observed. After nuclear staining with DAPI, the slides were mounted with ProLong gold antifade reagent (Thermo Fisher Scientific). The sections were observed and imaged by a Leica confocal microscope (Wetzlar, Germany).
6
Western Blot Analysis of Protein Expression
7
Immunohistochemical and Immunofluorescence Analysis
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For immunohistochemical detection, 5 μm paraffin sections were used; and endogenous biotin and peroxidase activity were blocked. The color was developed with DAB chromogen (ImmPACT DAB; VectorLabs, United States) and the sections were counterstained with hematoxylin.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.
Immunofluorescence analysis was performed as described22 (link). 6~8 μm frozen backskin sections were fixed in 4% PFA. For blocking, the MOM kit (Vector Laboratories, Burlingame, CA) was used for mouse monoclonal antibodies, and the following blocking buffer for all other antibodies: 10% normal donkey serum, 2% BSA, 2% fish skin gelatin, 2% Triton X-100 in PBS. Primary antibodies were used at the following concentration: guinea pig anti-Tcf3 (1:200, lab-generated), rabbit anti-Tcf4 (1:200; Cell Signalling), rabbit anti-Tcf1 (1:50; Cell Signaling), rabbit anti-Lef1 (1:150; Cell Signaling), mouse anti-myc tag (1:500; Zymed), mouse anti-β-catenin (Sigma), mouse anti-Stat3 (1:300; Cell Signaling), pY705-Stat3 (1:100; Cell Signaling), rat anti-cd104 (1:200; BD Biosciences) goat anti-Lcn2 (1:150;R&D Systems). Antigens were visualized with FITC-or RRX-conjugated secondary Abs (Jackson Labs). All images were acquired with Zeiss Axioskop microscope.
8
Renal Protein Quantification and Immunoblotting
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The total protein homogenate (30 μg) from the kidney was separated by sodium dodecyl sulfate-PAGE (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. Renal tissues were homogenized with ice-cold lysis buffer (mmol l -1 : Tris-HCl 20 (pH 7.5), EGTA 1, NaCl 150, β-glycerophosphate 1, sodium orthovanadate 1, Na2EDTA 1, PMSF 1, and sodium pyrophosphate 2.5, plus 1% Triton-X-100 and 1 μg ml -1 leupeptin). The signals were detected using enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA), and the images were analyzed using Image Quant LAS 4000 mini (GE Healthcare UK Ltd, Little Chalfont, UK). Here, the antibodies used were against rabbit anti-Smad3 (Cell Signaling Technology, Inc., Danvers, MA, USA; dilution 1:1000), rabbit anti-GAPDH (Cell Signaling Technology; dilution 1:1000), goat anti-LCN2 (R and D Systems, Minneapolis, MN, USA; dilution 1:1000) and mouse anti-β-actin (Sigma-Aldrich, St Louis, MO, USA; dilution 1:1000).
9
Cerebral Ischemia Tissue Protein Analysis
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After I/R 24h, the mice were killed by rapid cervical dislocation. The brain tissues were decapitated and were lysed by cell lysate. After standing for 1h, the tissues were centrifuged at 13200r/min for 20min. the collected supernatant was the cerebral ischemic tissue protein. BCA protein quanti cation kit was used to detect protein concentration. we performed SDS-PAGE gel electrophoresis with the same amount of protein mass (30mg), then transferred membrane(PVDF membrane). PVDF membranes were dissolved 5% skim milk in TBST and seal at room temperature for 2h, washed with TBST three times for 10min each time. Immunolabeling included primary antibody goat anti-LCN2 (1:500; R&D Systems), rat anti-Ly6G (1:500; Invitrogen), rabbit anti-occludin (1:1000; proteintech), rabbit anti-ZO-1 (1 :1000; proteintech), rabbit anti-CD206 (1:500; Abcam). PVDF membranes were incubated overnight at 4°C, washed three times with TBST at room temperature for 10min each time, then was applied secondary antibody, goat IgG secondary antibodies (1:1000; Millipore), rat IgG secondary antibodies (1:1000; Absin), rabbit IgG secondary antibodies (1:1000; Jackson).
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