Fibroblasts were seeded on 20- mm microscopy culture dishes at approximately 1×104 per well. Cells were fixed at room temperature for 20 minutes using 4% paraformaldehyde, then simultaneously blocked and permeabilized with a solution of 0.3% Triton X-100, 3% BSA, and 5% normal donkey serum (NDS) in phosphate-buffered saline (PBS) for 1 hour at room temperature. Cells were incubated with primary antibodies against IL-11 Rα (R&D Systems, Minneapolis, USA), α-SMA (Abcam, Waltham, USA), collagen type I α1 (COL1A1, Cell Signaling Technology, Beverly, USA) overnight at 4°C, and probed with the Alexa Fluor 488 or Alexa Fluor 546-labeled secondary antibody (both from Nanoprobes, New York, USA) in the dark at room temperature for 1 hour. Nuclei were visualized by staining with DAPI (Bioss, Beijing, China) for 10 min. Finally, an anti-fluorescence quenching agent (Solarbio, Beijing, China) was added, and samples were sealed and mounted. Samples were observed and imaged using a confocal microscope (Carl Zeiss 880, Oberkochen, Germany).
Il11rα
Manufactured by R&D Systems
Sourced in United States
IL11Rα is a protein that functions as a receptor for the cytokine interleukin-11. This receptor is important for the regulation of various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Startingblock blocking buffer, by Thermo Fisher Scientific (2 mentions)
Immobilon pvdf membrane, by Merck Group (2 mentions)
Bis tris nupage midi gel, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bioss Antibodies (1 mentions)
Anti fluorescence quenching agent, by Solarbio (1 mentions)
α sma, by Abcam (1 mentions)
Alexa fluor 488, by Nanoprobes (1 mentions)
Alexa fluor 546 labeled secondary antibody, by Nanoprobes (1 mentions)
Il 6rα, by R&D Systems (1 mentions)
4 protocols using il11rα
1
Immunofluorescent Staining of Fibroblasts
2
Western Blot Analysis of IL11Rα
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2×106 cells per sample were lysed in 100 μl of RIPA buffer. 10 μl of lysate was loaded per well of 4–12% Bis-Tris NuPage Midi gel (Life Technologies). Proteins were transferred to Immobilon PVDF membrane (EMD Millipore, Billerica, USA). Membranes were blocked for 30 minutes in StartingBlock blocking buffer (Thermo Scientific, Waltham, MA), then incubated overnight at 4°C with primary antibodies diluted 1:1000 in PBST in presence of 2.5% BSA. After 3×5 minute washes, membranes were incubated with secondary antibodies at 1:20,000 dilution, washed 2× 5 minutes followed by 20 minute wash. The membranes were developed with Immobilon substrate (EMD Millipore, Billerica, USA). The following antibodies were used: β-actin (Sigma, # A2228), IL11Rα (R&D systems #MAB1977), HRP conjugated anti-mouse and anti-rabbit (Thermo Scientific).
3
Western Blot Analysis of IL11Rα
4
ELISA Assay for Cytokine Receptor Binding
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We used an ELISA-based assay [71 (link)] to determine if MH can bind to recombinant IL-6Rα, IL-8R, and IL-11Rα proteins (all purchased from R&D Systems; Minneapolis, MN, USA). IL-6Rα (2 μg/mL), IL-8R (0.5 μg/mL), or IL-11Rα (1 μg/mL) proteins were coated on 96-well plates by overnight incubation at 4 °C. The plates were then washed with PBS + 0.05% tween 20 and blocked with PBS containing 1% BSA for 1 h at room temperature. For competition assay, various concentrations of MH (0.03–3%) were added to recombinant receptor-coated plates and incubated for 1 h at room temperature. After thorough washing, recombinant IL-6, IL-8, or IL-11 cytokines were added (50 ng/mL) and incubated for 1 h at 37 °C. The plates were then thoroughly washed and biotin-conjugated mAbs to IL-6 (0.25 µg/mL), IL-8 (0.5 µg/mL) or IL-11 (0.5 µg/mL) were added for 1 h at room temperature. After another round of washing, HRP-conjugated streptavidin was added for 30 min, washed, and developed by adding 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate for 10 min. Additionally, the potential effect of flavonoid compounds (luteolin, quercetin, galangin, and chrysin; dose range 0.5–50 μM) on binding to IL-6Rα was investigated in the same assay. The dose-response was modeled based on mass-action kinetic drug-response interactions.
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