Total proteins were extracted from colon tissue or kidney tissue samples when treated with radioimmunoprecipitation assay (RIPA) lysis (Sangon Biotech, Shanghai, China). The concentrations of total proteins were measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred to an NC membrane. The NC membrane was blocked with 5% non-fat milk and then incubated with primary antibodies against ZO-1 (AF5145, 1:500, Affinity), occludin (CY5997, Abways), claudin-1 (AF0127, 1:1,000, Affinity), Col-I (AF0127, 1:2,000, Affinity), Col-III (AF0136, 1:1,000, Affinity), FN (CY5621, 1:1,000, Abways), and LN (CY6617, 1:1,000, Abways) overnight at 4°C, respectively. Then, the membrane was washed with TBST and incubated with secondary goat anti-rabbit (S0001, 1:5,000, Affinity) and goat anti-rat antibodies (S0009, 1:5,000, Affinity) at 37°C for 2 h (Cell Signaling Technology, CA, USA). Finally, the proteins were monitored with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, NJ, USA) and then quantitatively analyzed by ImageJ software (version 1.4.0., National Institutes of Health). GAPDH protein was used for the internal control.
Af0136
Manufactured by Affinity Biosciences
Sourced in United States, China
The AF0136 is a specialized lab equipment designed for research and scientific applications. It serves as a core function in various laboratory processes, facilitating essential tasks required for scientific investigations. The detailed specifications and intended use of this product are not available within the scope of this request.
Automatically generated - may contain errors
Lab products found in correlation
Af7001, by Affinity Biosciences (6 mentions)
Af0127, by Affinity Biosciences (3 mentions)
Af1032, by Affinity Biosciences (3 mentions)
Af5145, by Affinity Biosciences (2 mentions)
Cy5997, by Abways (2 mentions)
Cy5621, by Abways (2 mentions)
Cy6617, by Abways (2 mentions)
Ab150077, by Abcam (2 mentions)
Bca assay, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Ripa lysis, by Sangon (1 mentions)
S0009, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
P38 mapk antibody, by Cell Signaling Technology (1 mentions)
Bleomycin sulfate, by Selleck Chemicals (1 mentions)
Sa00009 2, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
10 protocols using af0136
1
Quantitative Analysis of Extracellular Matrix Proteins
2
Histological Analysis of Cardiac Fibrosis
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A segment of the heart tissue was excised, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Serial 5-μm-thick sections were mounted and stained with hematoxylin and eosin (HE) and Masson's trichrome to examine the pathology of the heart. Furthermore, immune histology staining was performed for collagen I (1:200, AF7001, Affinity Biosciences, USA) and collagen III (1:200, AF0136, Affinity Biosciences, USA) in heart sections. Quantitative assessment of the fibrosis was determined based upon the extent of patchy and interstitial fibrosis. Each heart was observed, and at least 5 pictures were obtained in each region.
3
Peimine Fibrosis Inhibition Protocol
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Peimine (CAS: 23496-41-5) was prepared by Chengdu Munster Biotechnology Co., Ltd. (Chengdu, Sichuan, China). Bleomycin sulfate was obtained from Selleck (lot S1214) (Shanghai, China). Pirfenidone (PFD) capsules were obtained from the Beijing Kangdini Pharmaceutical Co. Ltd. (lot 150603) (Beijing, China). Antibodies against Collagen I, Collagen III, and CD68 were purchased from Affinity (AF7001, AF0136, DF7518), while antibodies against Arg-1, CD206, CTGF, TGF-β1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Proteintech (16001-1-AP, 60143-1-Ig, 23936-1-AP, 21898-1-AP, 60004-1-Ig). P-STAT6, STAT6, p-Akt (s473), Akt, p-p38 MAPK, p38 MAPK antibodies were obtained from Cell Signaling Technology (5397S, 56554S, 4060S, 4685S, 4511S, 8690S). FITC and Cy3-conjugated affinipure goat antirabbit IgG (H+L) were purchased from Proteintech (SA00003-2, SA00009-2).
4
Western Blot Analysis of Heart Tissue Proteins
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Protein lysates were harvested from heart tissues. The proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF, MilliporeSigma, Burlington, MA, USA) membranes. After being blocked with 5% non-at milk, the membranes were incubated with the following primary antibodies: anti-IFIT3 (1:1,000; A3924, ABclonal), anti-JNK (1:1,000; AF6318, Affinity, Changzhou, China), anti-p-JNK (1:1,000; AF3318, Affinity), p38 (1:1,000; AF6456, Affinity), p-p38 (1:1,000; AF4001, Affinity), ERK (1:1,000; AF0155, Affinity), p-ERK (1:1,000; AF1015, Affinity), collagen I (1:1,000; AF0134, Affinity), collagen III (1:1,000; AF0136, Affinity), α-SMA (1:1,000; AF1032, Affinity), and anti-β-actin (1:1,000; sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase (HRP) goat anti-rabbit IgG and HRP goat anti-mouse IgG (1:5,000; A0208/A0216, Beyotime) at 37°C for 45 min. Finally, the protein bands were visualized by using enhanced chemiluminescence liquid (ECL, Beyotime), and the OD values were analyzed by a Gel-Pro Analyzer (Liuyi, Beijing, China).
5
Histological Assessment of Renal Fibrosis
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The degree of renal tissue injury was evaluated by Masson’s trichrome staining. The kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4-μm paraffin sections. Then, sections were treated in xylene, dehydrated with graded ethanol, and stained with Masson (Sigma-Aldrich; Merck KGaA). After staining, the sections were dehydrated with 70 and 90% ethanol. Six fields of view were randomly selected and observed with an optical microscope (Olympus, Tokyo, Japan).
For immunohistochemistry (IHC) analysis, the renal tissues were incubated with the primary antibodies including anti-LN (CY6617, 1:100, Abways), anti-FN (CY5621, 1:100, Abways), anti-Col-I (AF0127, 1:200, Affinity), anti-Col-III (AF0136, 1:100, Affinity) overnight at 4 °C, respectively, and then further incubated with an anti-rabbit secondary antibody (ab150077, Abcam) for 2 h at room temperature. Finally, a representative area containing immunostained tissue was captured with a microscope (Olympus, Tokyo, Japan).
For immunohistochemistry (IHC) analysis, the renal tissues were incubated with the primary antibodies including anti-LN (CY6617, 1:100, Abways), anti-FN (CY5621, 1:100, Abways), anti-Col-I (AF0127, 1:200, Affinity), anti-Col-III (AF0136, 1:100, Affinity) overnight at 4 °C, respectively, and then further incubated with an anti-rabbit secondary antibody (ab150077, Abcam) for 2 h at room temperature. Finally, a representative area containing immunostained tissue was captured with a microscope (Olympus, Tokyo, Japan).
6
Kidney Tissue Protein Extraction and Western Blot Analysis
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Total protein was extracted from kidney tissues using RIPA buffer. 40 μg protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was transferred to the activated-PVDF membrane. After the block of antigen using 5% skimmed milk powder for 2 h at room temperature, the primary antibodies (CD9, Affinity, Df6565, 1:1000; CD63, Affinity, Df2306, 1:1000; CD81, Affinity, Df2305, 1:1000; α-SMA, Affinity, AF1032, 1:1000; collagen I, Affinity, AF7001, 1:1000; collagen III, Affinity, AF0136, 1:1000; Arginase1, Affinity, Df6657, 1:1000; iNOS, Affinity, Af6270, 1:1000; caspase3, Abcam, Ab184787, 1:1000; GAPDH, abcam, ab9485, 1:1000) were used to incubate the immunoblots overnight at 4 °C. The next day, the membrane was washed for 5–6 times (5 min/each) with TBST. Subsequently, the blots were incubated with HRP labeled second antibody (Goat Anti-Rabbit IgG (H + L) HRP, affinity, S0001, 1: 5000) in a shaker at 37 °C for 2 h. The immunoblots were visualized by adding ECL reagent and photographed by Gel imager. The results were analyzed by Image J software (National Institutes of Health, USA).
7
Comprehensive Protein Profiling for Cellular Analysis
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Collagen I antibody (AF7001, 1:1000), collagen III antibody (AF0136, 1:1000), α-SMA antibody (AF1032, 1:1000), p53 antibody (AF0879, 1:1000), p21 antibody (AF6290, 1:1000), and p16 antibody (AF5484, 1:1000) were purchased from Affinity Biosciences. IL11 antibody (A1902) was purchased from ABclonal. rmIL11 (Z03052) was purchased from GenScript, and L-KYN (L864410) was purchased from Macklin.
8
Evaluation of Oxidative Stress and Mitochondrial Dynamics
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Serum protein extraction kit (BB-319926, BestBio Science, Shanghai, China), Human ROS (reactive oxygen species) enzyme-linked immunoassay (Huijia Biotechnology Co., Ltd., Xiamen, China), SOD (superoxide dismutase) assay kit, and MDA (malondialdehyde) assay kit (A001-3-2, A003-1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used. Rabbit anti-MFN2 polyclonal antibody (sc-515647, Santa Cruz Biotechnology, Inc. USA), rabbit anti-Drp1 polyclonal antibody (8570S, Santa Cruz Biotechnology, Inc. USA), rabbit anti-OPA1 polyclonal antibody (67589S, Santa Cruz Biotechnology, Inc. USA), rabbit anti-Fis1 polyclonal antibody (sc-376446, Santa Cruz Biotechnology, Inc., USA), Rabbit anti-Col I (collagen type I) polyclonal antibody (AF7001, Affinity Biosciences, Inc., Jiangsu, China), rabbit anti-Col III (collagen type III) polyclonal antibody (AF0136, Affinity Biosciences, Inc., Jiangsu, China), rabbit anti-α-SMA (α-smooth muscle actin) polyclonal antibody (ab5694, Abcam Biotechnology, Inc., USA), and goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (ZB-2301, ZB-2305, Beijing Zhongshanjinqiao Biotechnology Co., Ltd., Beijing, China) were utilized.
9
Cardiac Proteins Quantification by Western Blot
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Total protein from the heart was extracted with ice-cold 1% Triton X-100 RIPA lysate supplemented with PMSF and then quantified by BCA. The protein was loaded in lanes, separated by SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with collagen I (1:1000, AF7001, Affinity Biosciences, USA), collagen III (1:1000, AF0136, Affinity Biosciences, USA), Scn5a antibody (1:1000, AF0255, Affinity Biosciences, USA) and GAPDH antibody (1:1000, D16H11, Cell Signaling Technology, USA) overnight at 4°C. Then, the membrane was incubated with HRP-labeled antibodies and exposed to HRP chemiluminescence. ImageJ 1.52 was used to quantify the bands, and GAPDH was used as a loading control to calculate the relative quantification among each group.
10
Quantitative Immunohistochemical Analysis
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For the immunohistochemistry (IHC) experiments, as above described, 4-µm-thick sections were prepared. The kidney tissues were incubated with antibodies against laminin (LN, CY6617, 1:100, Abways, Shanghai, China), fibronectin (FN, CY5621, 1:100, Abways), collagen I (Col-I, AF0127, 1:200, Affinity, Changzhou, China), and collagen III (Col-III, AF0136, 1:100, Affinity) overnight at 4°C. The colon tissues were incubated with antibodies against ZO-1 (AF5145, 1:100, Affinity), occludin (CY5997, Abways), and claudin-1 (AF0127, 1:200, Affinity) overnight at 4°C. Then, the tissues were incubated with an anti-rabbit secondary antibody (cat. no. ab150077; Abcam, Cambridge, USA) for 2 h at room temperature. Finally, a microscope (Olympus, Tokyo, Japan) was used to capture a representative area containing immunostained tissue (magnification, ×400). The staining intensity score was defined as follows: 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. The positive cell score was defined as follows: 0 = <5%, 1 = 5%–25%, 2 = 26%–50%, and 3 = >75%.
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