Aorta and kidney tissues were fixed in Histochoice (VWR, 12 h at 4 °C), rehydrated in 30 % sucrose/1XPBS (12 h at 4 °C), embedded in TFM (Fisher Scientific), and sectioned on the cryostat at 7 μm. Sections were prepared for immunofluorescent detection of either AT1 receptor (1:1000 dilution; #ab18801, Abcam, Cambridge, MA), AT2 receptor (1:1000 dilution; #ab19134, Abcam), MOMA-2 (1:1000 dilution; #ab33451, Abcam), renin (1:500 dilution; # AF4277, R&D Systems), or TNF-α (1:1000 dilution; #ab1793, Abcam) and/or von Willebrand factor (vWF -aorta sections only – 1:1000 dilution; #ab11713, Abcam), using anti-sheep Alexa Fluor 488 or anti-goat Alexa Fluor 647 and secondary antibodies (double the concentration of primary antibody used; Thermo Fisher Scientific). All slides were imaged and analyzed using Image J software (NIH, Bethesda, MD), as previously described by our laboratory [45 (link)]. Total fluorescence was quantified per unit area in kidneys and aortas; colocalization (endothelial cells of the aorta) was determined by quantifying the overlaid signals’ fluorescence from a minimum of 6 sections on 3 slides (n = 3–5 per group).
Ab18801
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab18801 is a lab equipment product offered by Abcam. It is a device designed for use in scientific research applications. The core function of this product is to perform a specific task or measurement, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab19134, by Abcam (2 mentions)
Ab33451, by Abcam (1 mentions)
Histochoice, by Avantor (1 mentions)
Ab11713, by Abcam (1 mentions)
Af4277, by R&D Systems (1 mentions)
Ab1793, by Abcam (1 mentions)
Anti nitrotyrosine, by Merck Group (1 mentions)
Western blotting reagents, by Merck Group (1 mentions)
Anti nox2, by Abcam (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Pa5 21690, by Thermo Fisher Scientific (1 mentions)
Ab89892, by Abcam (1 mentions)
Sc 8422, by Santa Cruz Biotechnology (1 mentions)
Sc 11360, by Santa Cruz Biotechnology (1 mentions)
Sc 30141, by Santa Cruz Biotechnology (1 mentions)
Sc 28879, by Santa Cruz Biotechnology (1 mentions)
Sc 377246, by Santa Cruz Biotechnology (1 mentions)
Ab24590, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Ab28379, by Abcam (1 mentions)
10 protocols using ab18801
1
Immunohistochemical Analysis of Aorta and Kidney Tissues
2
Protein Expression Analysis in Tissue
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NTS tissues were added with pre-cooled lysate and then cracked by ultrasonography. The specimens were statically placed on ice for 30 min to be fully cracked and then centrifuged at 12,000 rpm for 15 min at 4°CC. Supernatant was collected for protein assay. The Bradford assay (Generay Biotechnology, Shanghai) was used to determine the concentration of protein in tissue. An amount of 5 μg of protein was loaded in each lane. The protein was separated by electrophoresis, transferred onto polyvinylidene fluoride membranes, which were blocked with 0.1% Tween-20 tris-buffered saline containing 10% nonfat milk for 1 h at room temperature, and then incubated overnight at 4°C with the rabbit polyclonal antibodies anti-AT1R (1:800; ab18801, Abcam, USA), anti-ATRAP (1:500; sc-134652, Santa Cruz Biotechnology), anti-Nox2 (1:1,000; Abcam), anti-p67phox (1:1,000; EPITOMICS, USA), and anti-nitrotyrosine (1:1,000; Millipore), followed by the peroxidase-conjugated goat anti-rabbit secondary antibody (1:2,000; SA00001-2, Proteintech Biotechnology). Western blotting reagents (Millipore Corp., Billerica, MA) were used to detect the signal and blots were exposed to X-ray film for densitometric analysis.
3
Immunohistochemical Analysis of Kidney and Adrenal Tissues
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Kidney and adrenal gland tissues were fixed overnight in 4% formalin, dehydrated, and embedded in paraffin, using a conventional method. Paraffin-embedded samples were sectioned at a thickness of 3 μm. Sections were stained with hematoxylin and eosin (H&E) and trichrome. For immunohistochemistry, slides were incubated overnight with anti-AT1R or anti-Ang II antibodies at 4°C. The anti-renin antibody (PA5-21690) was obtained from Thermo Fisher (Waltham, MA, USA) and anti-AT1R (ab18801) and anti-Ang II (ab89892) antibodies were obtained from Abcam (Cambridge, UK). After staining, slides were examined using light microscopy.
4
Renal Expression Analysis of Key Proteins
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Renal expression of nephrin (1:1,000, sc-377246), fibronectin (1:1,000, sc-8422), type IV collagen (1:1,000, sc-11360), ACE (1:1,000, sc-23909), NOX4 (1:1,000, sc-30141), MCP-1 (1:2,000, sc-28879) (all from Santa Cruz Biotech, Santa Cruz, CA, USA), and AT1R (1:1,000, ab-18801) (Abcam, Cambridge, UK) was detected by Western blot analysis. Renal cortex tissues were homogenized in protein lysis buffer. All samples were centrifuged at 13,000 ×g for 20 minutes. The supernatants were mixed with the sample buffer and heated for 5 minutes at 95°C. Sample aliquots containing 15 to 30 μg of protein were loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (8% to 12%) and subsequently transferred to polyvinylidene difluoride membranes. The membranes were incubated at 4°C overnight with primary antibodies and washed three times in Tris-buffered saline with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The gels were visualized using an enhanced chemiluminescence gel electrophoresis system (Amersham Biosciences, Buckinghamshire, UK). The intensity of the bands was measured using ImageJ software version 1.50i (NIH, Bethesda, MD, USA).
5
Multiprotein Western Blot Analysis of Heart and Spleen Tissues
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Tissue protein of heart and spleen was lysed by the RIPA lysate (50:1) and tissue disrupter and quantified by the BCA method. The quantified homogenate was added to loading buffer, and the mixture was boiled and denatured at 99°C. Each lane was loaded with 10 ul of proteins. After electrophoresis at 100 V for 1–1.5 h, proteins were transferred to PVDF membranes at 300 mA for 1–1.5 h. Afterwards, the membrane blocked with 5% skim milk for 1–1.5 h at room temperature, incubated on a shaker, and washed with TBS-T. Western blot analysis was conducted using anti-AT1 (ab18801; Abcam, United States), anti-MCP-1 (ab25124, Abcam, United States), anti-CCR2 (PAI-27409), anti-TGF-β1 (3711s, Cell Signaling Technology, Germany), anti-Smad3 (ab28379, Abcam, United States), anti-MMP2 (ab86607, Abcam, United States), anti-Col III (ab7778, Abcam, United States), anti-VEGF (ab10972, Abcam, United States), anti-CD31 (ab24590, Abcam, United States) and anti-GAPDH (ab8245, Abcam, United States) at 4°C overnight. After incubation with the appropriate secondary antibodies at 37°C for 2 h at room temperature, the membrane treated with ECL for 1 min at room temperature. The final expression of each protein was normalized by GAPDH and grayscale analysis was performed using Image J software.
6
Western Blot Analysis of Signaling Proteins
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7
Immunofluorescence Staining Protocol
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Slides were prepared as described for Nile Red staining. 150 μl of the appropriate primary antibody: AGT (R&D Systems, Minneapolis, MN, #AF6996; 1:500), AT1 receptor (Abcam, Cambridge, MA; #ab18801, 1:1000), AT2 receptor (Abcam #ab19134; 1:1000), TNF-α (Abcam #ab1793; 1:1000), MCP-1 (Novus Biologicals, Centennial, CO; #NBP2−22115, 1:1000), GLUT4 (Novus Biologicals, NBP1−49533; 1:250), or IR-β (#NBP2−12793; 1:500) was applied for immunofluorescence (or double immunofluorescence) for 1 h at RT, followed by another incubation for 1 h at RT with the appropriate secondary antibody conjugated to either Alexa Fluor 455 or Alexa Fluor 555 (Thermo Fisher Scientific; double the concentration of primary antibody used), imaged (10x, 20x, 40x), and analyzed, as previously published [52 (link)]. Total fluorescence was quantified per unit area and normalized to total either total area or adipocyte cell count from a minimum of 2 slides, 4 sections, n = 3–5 per group.
8
Protein Expression Analysis in Cardiovascular Tissues
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Aorta, heart, and kidney homogenates were prepared in radioimmunoprecipitation assay lysis buffer (Wako, Tokyo, Japan). Samples were subjected to SDS‐PAGE. Proteins were transferred onto polyvinylidene fluoride microporous membranes (Bio Rad, Hercules, CA) and probed with primary antibodies, including monoclonal antibodies against BubR1 (Novus Biologicals, Littleton, CO; NBP1‐19555), Agtr1 (Abcam, Cambridge, UK; ab18801), Nox4 (Abcam; ab60940), JNK (Jun N‐terminal kinase; Abcam; ab9252), α‐tubulin (Abcam; ab4074), and Agtr2 (Santa Cruz Biotechnology; sc‐9040). HRP‐conjugated antirabbit IgG (1:5000, GE Healthcare UK, Buckinghamshire, UK; NA934V) was used as the secondary antibody. Chemi‐Lumi One Ultra (#11644; Nacalai Tesque, Kyoto, Japan) was used for chemiluminescence, and an Amersham Imager 600 (GE Healthcare UK) was used for imaging.
9
Protein Expression Analysis in Myocardial Tissue
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Fifty micrograms of cell lysates and myocardial tissue were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto nitrocellulose membranes. Anti-Angiotensin converting enzyme 1 antibody (ACE) (ab216476) and anti-Angiotensin II type 1 receptor antibody (AT1R) (ab18801) were purchased from Abcam, Cambridge, UK. HRP conjugated immunoglobulin was used as a secondary antibody (Jackson ImmunoResearch Laboratories). West Pico Chemiluminescent (Pierce) was used as the substrate to visualize protein bands and quantified using densitometry image analysis software (Image Master VDS; Pharmacia Biotech).
10
Quantifying Kidney Protein Levels
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Protein concentrations of whole kidney samples were determined using the BCA protein assay kit (Thermo Fisher, Belgium) . Samples containing the same amount of proteins were separated on a 12% SDS-page gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Belgium), then transferred to a polyvinylidene fluoride membrane and blocked 2h with 5% bovine serum albumin (BSA) in Tris-buffered solution containing 0.1% Tween-20 (TBS-T). To investigate whole kidney RAAS protein expression, the membrane was incubated overnight at 4°C in the presence of an antiangiotensin II type I receptor antibody (ATIIT1R, 1/2000, ab18801, Abcam, UK), as previously described (Wang et al., 2015) . Secondary swine anti-rabbit horseradish peroxidase-conjugated antibody (P0217, DAKO, Belgium) at a dilution of 1/2500 was used. Both primary and secondary antibodies were diluted in BSA-TBS-T. ATIIT1R was visualized using the chemiluminescence (ECL) technique (1 min exposure) using the Pierce ECL Plus Western Blotting Substrate Kit (Thermo Fisher, Belgium) and quantified using Image Quant TL software v8.1 (GE Healthcare Europe, Belgium). Data were normalized to β-actin protein levels (1/2500, sc-4778, Santa Cruz, USA) (Cops et al., 2018a; Cops et al., 2018b ) (figure S1).
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