The renal tissue samples from all animals were fixed with 4% paraformaldehyde for 3 days and embedded in paraffin. The 5 μm sections were stained with haematoxylin and eosin (H&E), specific antibodies and Masson's trichrome for histological evaluation and scoring. The protein signal was detected using the appropriate primary antibody amplifier, horseradish peroxidase (HRP)‐conjugated polymer and DAB chromogen/substrate. The slides were immunostained with anti‐NFkB p65 (50‐fold of dilution; Abcam), anti‐ALPK1 (100‐fold of dilution; GeneTex Inc), anti‐CCL2 (50‐fold of dilution; Abcam) and anti‐CCL5 (50‐fold of dilution; Abcam). The tubular injury was scored based on the degree of tubular necrosis, loss of brush border, cast formation and tubular dilatation, as in previous reports.19 , 20 The scoring standard was as follows: 0‐normal kidney; 1‐minimal injury (<5% involvement); 2‐mild injury (5%‐25% involvement); 3‐moderate injury (25%‐75% involvement); and 4‐severe injury (>75% involvement).
Anti ccl2
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-CCL2 is a laboratory reagent used to detect and quantify the chemokine CCL2 (also known as MCP-1) in biological samples. It is a highly specific and sensitive antibody that can be used in various immunoassay techniques to measure CCL2 levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (4 mentions)
Anti ccr2, by Abcam (3 mentions)
Anti ccl5, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti cd68, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Bovine serum, by Bio-Rad (2 mentions)
Anti alpk1, by GeneTex (1 mentions)
Anti nf kb p65, by Abcam (1 mentions)
Anti bip, by Abcam (1 mentions)
Anti chop, by Abcam (1 mentions)
Anti manf, by Abcam (1 mentions)
Anti ho 1, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Anti hmgb1, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
16 protocols using anti ccl2
1
Histological Evaluation of Renal Injury
2
Molecular Mechanisms of Neuroinflammation
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Antibodies involved in this study contain: anti-Cleaved caspase3 (Abcam, ab32042); anti-HMGB1 (Abcam, ab79823); anti-TNF-α (Abcam, ab183218); anti-IL-1β (Abcam, ab9722); anti-CCL2 (Abcam, ab25124); anti-Bip (Abcam, ab21685); anti-CHOP (Abcam, ab11419); anti-HO-1 (Abcam, ab52947); anti-MANF (Abcam, ab67271); anti-GAPDH (Abcam, ab3285); anti-HIF-1α (Abcam, ab243860); Anti-CD163 (Abcam, ab182422); Goat Anti-Rabbit IgG H&L (HRP) (Abcam, ab6721); PE anti-CD11b (Abcam, ab25533); APC anti-Ly6C (Abcam, ab93550); Alexa Flour 488 anti-Ly6G (Abcam, ab283276). The involved reagents contain: DNFB (Sigma, St Louis, MO, USA, 42085); hrMANF protein (Abcam, ab123227); Goat Anti-Mouse/Rabbit Polymer Immunohistochemistry Detection Kit (ZSGB-BIO, PV-6000); Lipofectamine™ 3000 (Thermo Fisher, L3000150).
3
Immunohistochemical Analysis of CCL-2, CCL-5, α-SMA, and Collagen IV
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Immunohistochemistry was performed as previously described in paraffin-embedded tissue sections at a thickness of 4 µm [18] (link). The primary antibodies used in this study were as follows: anti-CCL-2 (1∶200, Abcam), anti-CCL-5 (1∶100, Santa Cruz), anti-α-SMA (1∶100, Sigma) and anti-collagen IV (1∶400, Abcam). Sections were counterstained with hematoxylin. Positive staining were quantified by Image Pro Plus Software 5.0 (Media Cybernetics, Silver Spring, USA) and presented as IOD value.
4
Quantitative Analysis of CX3CL1 and CCL2 Proteins
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Tissue samples from STANR, AF, and NP were rapidly removed and stored at −80°C until use. All tissue samples were homogenized in lysis buffer containing phenylmethane sulfonyl fluoride and 0.02% protease inhibitor cocktail. The homogenates were centrifuged at 14,000×g for 15 min at 4°C. Equivalent amounts of protein (40 μg) were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk for 1 h at room temperature and incubated with respective primary antibodies anti-CX3CL1 (1:1,000; Santa Cruz Biotechnologies, Inc) and anti-CCL2 (1:4,000; Abcam, Cambridge, UK) overnight at 4°C. The membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:5,000; Santa Cruz Biotechnologies, Inc). Bands were finally revealed using an enhanced chemiluminescence kit (Kodak, Rochester, NY, USA). The monoclonal antibody against β-actin (1:10,000; Sigma-Aldrich Co., St Louis, MO, USA) was used as a loading control. Bands area/intensity for all proteins were measured using Image J software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA). All the samples were routinely analyzed using Western blot repeated for at least three times, and the obtained results were averaged and calculated.
5
SDS-PAGE and Immunoblotting of Cell Proteins
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Total cell protein was separated on 10% SDS-PAGE (Pall Corporation, USA), and transfected to the membranes (EMD Millipore, USA). Then, the membranes were blocked with 5% BSA for 1 h and immunoblotted with anti-CCL2 (1:500, affinity, USA), anti-CCR2 (1:500, affinity, USA), anti-GDNF (1:500, Abcam, USA), anti-p-RET (1:500, affinity, USA), anti-RET (1:500, affinity, USA), or anti-β-actin (1:1,000, affinity, USA). The results were visualized with a chemiluminescence reagent (Thermo, USA).
6
Molecular Mechanisms of Pentobarbital-Induced Necroptosis
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Pentobarbital sodium was safeguarded in Harbin Medical Pharmacological Laboratory. BSA and rhTrx‐1 were purchased from R&D System. Penicillin/streptomycin solution, Hoechst and 2% TTC dyeing solution were purchased from Solarbio. ROS and Necrostatin‐1 was purchased from Sigma. Annexin V‐PE/7AAD assay was purchased from BD Biosciences. All the ELISA kits were purchased from Cusabio. Dapi dyeing solution and JC‐1 kit were purchased from Beyotime. DMEM, foetal bovine serum (FBS) and MEM/EBSS were purchased from Hyclone. The primary antibodies used for immunofluorescence staining: Rabbit polyclonal anti‐Iba‐1 (Wako), Goat polyclonal anti‐Iba‐1 (Abcam), Mouse monoclonal anti‐RIPK1 (Santa cruz), Goat polyclonal anti‐CD206 (R&D System) and Rabbit polyclonal anti‐CD16 (Bioss). All the second antibodies used for immunofluorescence staining were purchased from Abcam company. The primary antibodies used for Western blot analysis: anti‐RIPK1, anti‐RIPK3, anti‐MLKL, anti‐pMLKL and anti‐CCL2 (Abcam); anti‐NLRP3, anti‐ASC, anti‐caspase‐1, anti‐caspase‐3 and anti‐β‐actin (ABclonal); anti‐MMP‐9 (R&D System); and anti‐CD16 (Bioss). The second antibodies used or Western blot analysis was Alexa Fluor 800‐conjugated Goat‐anti rabbit (LI‐COR).
7
Immunoblotting Analysis of Chemokine Signaling
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Cultured cells were lysed on ice with 100 µl lysis buffer, collected by a cell scraper for a violent vortex, and then centrifuged at 4°C at 12,000 rpm for 10 min. Supernatants were collected, and the quantified proteins were boiled for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Samples were run on 6 and 12% Tris-Glycine gels separately. Immunoblotting was performed according to standard protocol, transferred to polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA), blocked with tris buffered saline with tween (TBST) + 5% skim milk for 2 h at room temperature, incubated overnight at 4 °C with the following antibodies: anti-CCL2 (Abcam, #9669, Cambridge, MA, USA), anti-β-actin (Abcam, #8226), and anti-CXCL10 (Abcam, #9807). After incubating with the aforementioned antibodies, samples were washed three times with horseradish peroxidase and incubated with HRP-conjugated IgG antibodies for 2 h at room temperature. Specific bands were detected using the chemiluminescent substrate (Thermo Fisher, Waltham, MA, USA). Immunoblot bands were quantified in relation to β-actin using Image-Pro Plus.
8
Protein Extraction and Immunoblotting
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Proteins were extracted with RIPA lysis buffer and quantified by the BCA kit (Roche). The proteins were separated by SDS/10% PAGE and transferred to a PVDF membrane for immunoblotting assay with the antibodies [Anti-GAPDH (#2118, Cell Signaling Technology) and Anti-CCL2 (ab8101, Abcam)].
9
Western Blot Analysis of HUVECs
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HUVECs were collected using RIPA buffer (Beyotime, Shanghai, China) and were sonicated at 4°C for 30 s after it was incubated on ice for 30 min. The supernatant was collected after centrifuging at 12 000g for 10 min at 4°C for BCA protein quantification (Beyotime, Shanghai, China). Western blot analysis was performed as described previously. Primary polyclonal anti-CCL2 (1 : 1000), anti-NOX4 (1 : 2000), anti-PPARα (1 : 2000), anti-PDGFβ (1 : 1000), anti-VWF (1 : 2000) and anti-β-actin (1 : 5000) antibodies were all purchased from Abcam (Abcam, MA, USA). The primary antibody was added to the PVDF membrane for incubation overnight at 4°C; after the membrane was washed three times for 15 min with TBST at room temperature (RT), it was incubated with a secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse immunoglobulin (Ig)G antibody (1 : 2000; CST, MA, USA) for 2 h at RT. The western blot bands were visualized using the enhanced chemiluminescence kit (Beyotime, Shanghai, China) and quantified via densitometric analysis using Image -Pro Plus v. 6.0 software (Media Cybernetics, USA).
10
Antibody Characterization Protocol
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The following antibodies were used: anti-LYVE-1, Abcam (ab14917), for IHC; anti-F4/80, Abcam (ab6640), for IHC; anti-hnRNPL, Abcam (ab6106), for immunoblot, RIP and ChIP; anti-hnRNPQ, Abcam (ab184946), for immunoblot; anti-hnRNPA1, Abcam (ab4791), for immunoblot; anti-hnRNPL, Abcam (ab133607), for immunoblot; anti-CD68, Cell Signaling Technology, #76437, for IHC and flow cytometry; anti-CD206, Abcam (ab64693), for flow cytometry; anti-CCL2, Abcam (ab9669), for immunoblot and IHC; CCL2-neutralizing antibody (#554440, BD Biosciences, NJ, USA); anti-GAPDH, Cell Signaling Technology, #5174, for immunoblot; anti-H3K4me3, Abcam (ab8580), for immunoblot and ChIP; anti-VEGF-C, Abcam (ab9546), for immunoblot and IHC; and VEGF-C-neutralizing antibody (pV1006R-r, Angio-Proteomie, Boston, MA, USA). Control mouse IgG, control rabbit IgG, anti-RNA pol II, and anti-snRNP70 were provided in the EZ-Magna RIP kit or EZ-Magna ChIP A/G kit (Millipore). DAPI (Thermo Scientific, 62247) and Alexa Fluor™ 555 Phalloidin (Invitrogen™) were also used.
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