The enucleated eyes from each mouse were lysed and the total proteins were extracted as previously described (20 (link)), and the protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins (40 µg) in each sample were loaded and separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat milk overnight and probed with anti-p35 (ab66064), anti-IL-10 (ab134742), anti-TFG-β (ab190503), anti-IFN-γ (ab24324), anti-IL-12 (ab89889) and anti-IL-17 (ab77171) primary antibodies (1:500 dilution; all from Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with secondary anti-rabbit IgG-HRP antibody (A0208) or anti-mouse IgG-HRP antibody (A0216) (1:5000 dilution; Beyotime Institute of Biotechnology) at 37°C for 45 min after washing twice with TBST. The target bands were visualized using enhanced chemiluminescence (ECL) solution (Qihai Biotec, Shanghai, China) and analyzed using Gel-Pro-Analyzer software (Media Cybernetics, Bethesda, MD, USA). β-actin served as an internal control.
Ab134742
Manufactured by Abcam
Sourced in United States
Ab134742 is a laboratory equipment product. It is a compact and versatile device designed for general laboratory use. The core function of this product is to perform a specific task in the laboratory setting. No further details are available to provide an unbiased and factual description while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Gel pro analyzer software, by Media Cybernetics (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Ab213612, by Abcam (1 mentions)
Sc 58986, by Santa Cruz Biotechnology (1 mentions)
Ripa lysate, by Merck Group (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab2787, by Abcam (1 mentions)
Ab119992, by Abcam (1 mentions)
Ab267787, by Abcam (1 mentions)
Ab20346, by Abcam (1 mentions)
Ab32089, by Abcam (1 mentions)
Ab108424, by Abcam (1 mentions)
Ab92486, by Abcam (1 mentions)
Zk 9600, by OriGene (1 mentions)
Pv 6000, by OriGene (1 mentions)
4 protocols using ab134742
1
Western Blot Analysis of Ocular Proteins
2
Western Blot Analysis of Exosome Proteins
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The cells or exosomes were lysed with RIPA lysate (Sigma) to obtain proteins. The BCA Assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. An equal amount of protein was added onto 10% SDS-PAGE gels, and the protein was then transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The blots were then incubated with primary antibodies, Anti-PI3Kγ antibody (1 : 1,000, ab32089, Abcam), Anti-CD163 antibody (1 : 1,000, ab213612, Abcam), Anti-CD206 antibody (1 : 1,000, sc-58986, Santa Cruz Biotechnology), Anti-PTEN antibody (1 : 1,000, ab267787, Abcam), Anti-AKT antibody (1 : 1,000, ab213612, Abcam), Anti-pAKT antibody (1 : 1,000, ab38449, Abcam), Anti-Vimentin antibody (1 : 1,000, ab20346, Abcam), Anti-α-SMA antibody (1 : 1,000, ab108424, Abcam), Anti-IL-10 antibody (0.5 µg/ml, ab134742, Abcam), Anti-CD63 antibody (1 µg/ml, ab38418, Abcam), Anti-CD61 antibody (1 : 1,000, ab119992, Abcam), Anti-HSP70 antibody (1 : 1,000, ab2787, Abcam), and anti-Actin (1 µg/ml, ab8286, Abcam) overnight at 4°C. The horseradish peroxidase-conjugated secondary antibody was then added to incubate the blots at room temperature for 2 h. At last, the blots were visualized with the Novex™ chemiluminescent substrate reagent kit (Waltham, MA, USA).
3
Immunohistochemical Analysis of IL-10 and IL-17
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Paraffin sections were dewaxed, hydrated, and blocked with 3% H2O2 for 15 minutes to inactivate endogenous peroxidase. Antigens were retrieved with 10 mM sodium citrate buffer. After three washes with PBS, samples were blocked with goat serum for 30 minutes and incubated with anti-IL-10 (Abcam, ab134742) and anti-IL-17 (Abcam, ab92486) at 4°C overnight. After three washes with PBS, secondary antibody was added and incubated at 37°C for 2 hours. Nuclei were counterstained with hematoxylin. Samples were visualized with 3,3′-diaminobenzidine, mounted with neutral resin, and observed under the light microscope.
4
Quantifying Intestinal IL-10 Protein Expression
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Immunohistochemistry was carried out to assess IL-10 protein expression in the mucosa biopsy tissues from distal ileum and sigmoid. Sections were deparaffinized in xylene and rehydrated in decreasing concentration of ethanol (100%, 95%, 80%) and subjected to immunohistochemical technique using the ZSGB-BIO ALK system (ZK-9600; Origene; and ZSGB-BIO; MO BIO, Beijing, China) After antigen retrieved and endogenous peroxidases blocked, primary antibody against IL-10 (ab134742, 1:800; Abcam, Cambridge, MA, USA) was incubated for 12 h at 1:800 dilution. As a secondary antibody and for visualization, a peroxidase/3, 3′-diaminobenzidine-positive was used according to the manufacturer's protocol (ZSGB-BIO ALK Detection System Peroxidase 3, 3′-diaminobenzidine-positive mice; PV-6000; Origene; and ZSGB-BIO; MO BIO). The IL-10 levels were evaluated based on integral optical density of positive stain sing Image Pro Plus 6.0.
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