IL-6Rα expression (ab83053, Abcam) was evaluated in discoidin domain receptor 2+ (DDR2; ab76967, Abcam) CFs using immunohistochemistry. The influence of IL-6 on murine specific matrix metalloproteinase 9 (MMP-9) was quantified using media conditioned by murine CFs after 48 h of 1% oxygen and 1% serum direct co-culture with SCR or shIL-6-transduced human EDCs (MMPT90, R&D Systems). The effect of IL-6 on CFs and cardiac macrophages in vivo was assessed 28 days post-infarct using histological analysis of myocardial sections for alpha smooth muscle actin (αSMA; ab66133, Abcam), the myofibroblast marker CD68 (ab955, Abcam) or M2 macrophage marker CD206 (ab64693, Abcam).
Ab66133
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab66133 is a monoclonal antibody that can be used to detect the presence of a specific target protein in biological samples. It is designed for research use only and its core function is to provide a reliable and consistent method for target protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab76967, by Abcam (1 mentions)
Ab83053, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Mmpt90, by R&D Systems (1 mentions)
Ab955, by Abcam (1 mentions)
Paraffin, by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
Sc 146, by Santa Cruz Biotechnology (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Sc 20157, by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i 90i, by Nikon (1 mentions)
Ab76533, by Abcam (1 mentions)
A6003, by Merck Group (1 mentions)
S7900, by Merck Group (1 mentions)
E7889, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Ab124762, by Abcam (1 mentions)
Ab53115, by Abcam (1 mentions)
Ab124679, by Abcam (1 mentions)
Biotin conjugated secondary antibody, by Vector Laboratories (1 mentions)
6 protocols using ab66133
1
Regulation of Cardiac Fibrosis by IL-6
2
Histological Analysis of Cardiac Fibrosis
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All the mice were sacrificed on day 7 with pentobarbital, and the hearts were collected, washed with a heparin-containing saline solution (Sigma-Aldrich), fixed in 10% formalin (Sigma-Aldrich), and embedded in paraffin (Sigma-Aldrich). The paraffin-embedded fixed tissues were then cut into 5-µm-thick sections, placed on polylysine-coated glass slides (HL-H05-2; Nantong Hailun Bio-Medical Apparatus Manufacturing Co., Ltd., Haimen, China) and stained with hematoxylin (Sigma-Aldrich) and Masson's trichrome reagent (Beijin Solarbio Science & Technology Co., Ltd.). The fibrotic areas were quantitated as the ratio of the area that had stained blue to the total section area using the NIS-Elements analysis program (Nikon Corporation, Tokyo, Japan). Immunohistochemistry was performed using a standard procedure as previously described (2 (link)). The sections were incubated overnight at 4°C with antibodies against transforming growth factor β1 (TGF-β1; rabbit polyclonal; sc-146; 1:200; Santa Cruz Biotechnology, Inc., CA, USA), α-smooth muscle actin (α-SMA; rabbit polyclonal; ab66133; 1:300; Abcam, Cambridge, MA, USA), and Mac-2 (galectin-3; rabbit polyclonal; sc-20157; 1:200; Santa Cruz Biotechnology, Inc.). The images were captured using a microscope equipped with a camera (ECLIPSE 80i/90i, Nikon Corporation).
3
Immunofluorescent Characterization of VSMCs
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VSMC cultures were trypsinized at cell density of 200,000 cells/5 mL in T25 flasks and then fixed with 4% paraformaldehyde during 10 min at room temperature. VSMCs were permeabilized with 0.5% saponin (S7900, Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 5 min and blocked with 1% BSA (A6003, Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Primary antibodies used were anti-alpha smooth muscle actin at 1/100 (ab66133, Abcam, Cambridge, UK), transgelin/SM22 monoclonal antibody at 1/100 (60213-1-Ig, ProteinTech, Manchester, UK) and anti-CD31 (also called platelet and endothelial cell adhesion molecule 1, PECAM1) [EPR3094] antibody at 1/50 (ab76533, Abcam, Cambridge, UK) during 1 h in shaking at room temperature. VSMCs were washed with 5 mM EDTA (E7889, Sigma-Aldrich, St. Louis, MO, USA) and 0.5% BSA in PBS and were incubated with secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG (H + L) (A-11001, Invitrogen, Thermofisher, Carlsbad, CA, USA) and Alex Fluor® 647 goat anti-rabbit IgG (H + L) (A-21244, Invitrogen, Thermofisher, Carlsbad, CA, USA) at 1/500 for 30 min in shaking and darkness. After washing, cells were resuspended in 5 mM EDTA and 0.5% BSA in PBS.
4
Immunohistochemical Characterization of Cells
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Fresh sections (formalin-fixed, paraffin embedded) were dewaxed following standard protocols and antigen retrieval with pH6 citrate retrieval buffer was undertaken for SMMHC, CK14, p63 and Ki67 staining. Sections were stained with anti-α-smooth muscle actin (1 μg/ml, Abcam, ab66133), anti-smooth muscle myosin heavy chain 1+2 (1 μg/ml, Abcam, ab124679), anti-Ki67 (1 μg/ml, Abcam, ab15580), anti-cytokeratin 14 (5 μg/ml, Abcam, ab53115), anti-p63 (0.75 μg/ml, Abcam, ab124762), or with isotype control antibodies, overnight at 4°C. Biotin-conjugated secondary antibodies (Vector Laboratories, CA, USA) were applied for 1 h and signal was amplified by conjugation of HRP (ABC kit, Vector Laboratories). Antibodies were visualized with DAB prior to nuclear counterstaining with hematoxylin.
5
Cardiac Differentiation of EDCs
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The effect of variable culture conditions on the ability of EDCs to adopt a cardiac phenotype was assessed by plating 20,000 cells/cm2 within cardiogenic media [9 , 21 (link), 26 ]. Cardiogenic media consisted of low glucose Dulbecco’s modified Eagle’s media, MCDB-201 media, dimethylsulfoxide, l -ascorbic acid, 0.01% insulin-transferrin-selenium liquid media supplement, linoleic acid-albumin, penicillin-streptomyin, dexamethasone, 2-mercaptoethanol, recombinant human fibroblast growth factor 8b, recombinant human fibroblast growth factor 4, recombinant human protein Dickkopf-related protein 1, and recombinant human bone morphogenetic protein 2 (all from Life Technologies) [27 (link)]. After 7 days of culture, cells were harvested for flow cytometry (alpha smooth muscle actin (α-SMA; ab125266, Abcam), cardiac troponin T (cTnT; ab66133, Abcam), or von Willebrand factor (vWF; 11778-1-AP, ProteinTech)).
6
Immunofluorescence Analysis of α-SMA
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Sections were rehydrated and permeabilized with 1% triton-X for 20 minutes. Nonspecific binding was blocked by placing the sections in 6% BSA for 4 h in a humidified chamber. Samples were washed three times in PBST containing 2% BSA for 5 min, incubated overnight in a humidified chamber with α-SMA primary antibody (1:50, Abcam, ab66133), and washed three times with PBST containing 2% BSA for 5 min. The slides were incubated in a humidified chamber with an anti-rabbit secondary antibody tagged with Alexa Fluor (1:100, A10042; Invitrogen, Carlsbad, CA) for 2 h, and washed three times with PBST containing 2% BSA for 5 min. The sections were stained with DAPI (4′–6-diamidino-2-phenylindole). The immune complexes were examined under a fluorescence microscope (Leica DM 2500; Leica) and imaged.
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