Cell lysates were extracted using RIPA buffer (CW Biotech Co. Ltd., Beijing, China). The BCA protein assay kit (CW Biotech Co. Ltd., Beijing, China) was performed to measure the protein concentration. Equal amounts of proteins were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene fluoride (PVDF) membranes and incubated with primary antibodies, including anti-SYK antibody (ab40781) (Abcam, Cambridge, MA, USA), anti-collagen I antibody (ab34710) (Abcam, Cambridge, MA, USA), anti-vimentin antibody (ab8978) (Abcam, Cambridge, MA, USA), anti-VEGF-A antibody (ab46154) (Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (ab1416) (Abcam, Cambridge, MA, USA), anti-α-SMA antibody, (ab32575) (Abcam, Cambridge, MA, USA), anti-TGF-β1 antibody (ab64715) (Abcam, Cambridge, MA, USA), anti-p-Smad3 antibody (ab52903) (Abcam, Cambridge, MA, USA), and anti-Smad3 antibody (ab40854) (Abcam, Cambridge, MA, USA). The membranes were then incubated in the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1/1000) (ab6721) (Abcam, Cambridge, MA, USA) or anti-mouse immunoglobulin G secondary antibody (1/1000) (ab6785) (Abcam, Cambridge, MA, USA). The results were analyzed using the enhanced chemiluminescence (ECL) Western blot substrate (Pierce Biotechnology, Shanghai, China). GAPDH was used as an internal control.
Ab64715
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab64715 is a recombinant protein product. It is produced using a baculovirus expression system.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (4 mentions)
Ab9722, by Abcam (3 mentions)
Ab6992, by Abcam (3 mentions)
Ab76055, by Abcam (3 mentions)
Ab53100, by Abcam (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab3523, by Abcam (2 mentions)
Ab63672, by Abcam (2 mentions)
3 3 diaminobenzidine tetrachloride dab, by Zymo Research (2 mentions)
Ab7778, by Abcam (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ab62623, by Abcam (2 mentions)
Vectastain abc hrp kit, by Vector Laboratories (2 mentions)
Ab28052, by Abcam (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab52903, by Abcam (1 mentions)
36 protocols using ab64715
1
Protein Expression Analysis in Cellular Lysates
2
Comprehensive Antibody Panel for Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).
3
Quantification of Hepatic TGF-β1 and Angiogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The liver was fixed in 10% neutral buffered formalin, then immunostained with anti-TGF-β1 antibody (ab64715, Abcam, Tokyo, Japan). The number of TGF-β1-positive hepatic cells was counted in 10 randomly selected high-power microscopic fields (×400), and the expression rate of TGF-β1 was calculated by dividing the number of TGF-β1-positive hepatic cells by the number of all hepatic cells in a high-power microscopic field (×400). The quantified expression rate of TGF-β1 was averaged for each group, and results are expressed as mean value ± standard error of the mean.
The liver was fixed in zinc solution, then immunostained with anti-CD31 antibody (sc-1506, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). The number of CD31-positive vessels/field was quantified by averaging the number of CD31-positive vessels in 10 randomly selected high-power microscopic fields (×400). The results are expressed as mean value ± standard error of the mean.
The liver was fixed in zinc solution, then immunostained with anti-CD31 antibody (sc-1506, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). The number of CD31-positive vessels/field was quantified by averaging the number of CD31-positive vessels in 10 randomly selected high-power microscopic fields (×400). The results are expressed as mean value ± standard error of the mean.
4
Immortalized Stromal Cell Coculture Protocol
5
Renal Fibrosis Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Take 50 mg renal tissue from liquid nitrogen, according to the appropriate ratio, PMSF, RIPA, and Phosphatase Inhibitors were added to the centrifuge tube which contained renal samples. 14,000 r/min for 15 minutes at 4°C, BCA method to determine the protein concentration. Protein lysates were separated by 10% SDS-PAGE, then transferred to PVDF membranes (Millipore, USA), and blocked with 5% nonfat milk for one hour. The PVDF membranes were incubated overnight with primary antibodies. The primary antibodies were TGF-β1 (1:300; abcam, ab64715), Smad2/3 (1:1000; abcam, ab63672), Smad7 (1:3000; abcam, ab190987), CTGF (1:1000; abcam, ab6992), Collagen I (1:1000; abcam, ab34710), Collagen III (1:5000; abcam, ab7778), and GAPDH (1:5000; weiao, shanghai). The next day, membranes were washed by TBST for three times. After that, the membranes were incubated with secondary antibodies for one hour at room temperature. After another three times washed by TBST, the signal was exposed on ProteinSimple FluorChem M and used IMAGE J software to analyze the gray value.
6
Fluorescent Antibody Labeling for In Vivo Use
7
Tregs Isolation and Conditioned Medium Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CD4+ CD25+ Regulatory T Cell Isolation Kit (ID: 130-091-041, Miltenyi, Bergisch Gladbach, Germany) was used to isolate Tregs from the spleen of mice. Tregs were cultured in RPIM-1640 (Gibco-BRL, Gaithersburg, MD, USA) with 10% FBS for 24 hours, then changed to serum-free medium for 24 hours. The supernatant of Tregs-conditioned medium (Tregs-CM) was collected by centrifugation. Tregs-CM was incubated with 1 µg/mL TGF-β1-neutralizing antibody (mouse monoclonal anti-TGF-β1, [9016] (ab64715); Abcam) for 24 hours at 4°C to obtain TGF-β1-depleted Tregs-CM. A total of 5×105 HCC Hepa1–6 cells/well (Shanghai Cell Bank of the Chinese Academy of Sciences) were precultured in 2 mL DMEM (Gibco-BRL) with 10% FBS in 6-well plates for 24 hours. Then, the culture medium was replaced with 1 mL Tregs-CM and 1 mL complete medium per well for 24 hours. The treated Hepa1–6 cells were used in the subsequent experiment.
8
Macrophage Polarization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophages were obtained from bone marrow precursor cells. In brief, bone marrow was obtained from mice by flushing the femur and tibia with DMEM. Cells were cultured for 7 d in DMEM conditioned by L929 cells (enriched in CSF-1). Macrophages were polarized toward a proinflammatory phenotype with 50 ng/ml IFN-γ in DMEM containing 10% FBS and were incubated with or without 40 nM fr-HMGB1 or 3S for 3 d. Macrophages were labeled with primary antibodies against iNOS (ab3523; Abcam), TNFα (ab34839; Abcam), CD163 (sc-33560; Santa Cruz), TGFβ1 (ab64715, Abcam), and Cy3- or FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).
9
Probing Intestinal Epithelial TGF-β Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sixty micrograms of protein extracted from the isolated rat intestinal epithelial tissues samples was separated by SDS-PAGE and transferred to a PVDF membrane. Blots were blocked for 1 h in 5 % BSA and then incubated with primary antibodies for overnight at room temperature [4 (link)]. Primary antibodies were directed against TGFβ1 (1:500) (Ab64715, Abcam), TβR1 (1:750) (Ab31013, Abcam), TβR2 (1:500) (Ab186838, Abcam), smad3 (1:1000) (#9523, CST), Snail (1:1000) (Ab180714, Abcam), E-cadherin (1:1000) (Ab76055, Abcam), and Fibronectin (1:1500) (Ab6328, Abcam). The films were visualized using an enhanced chemiluminescence system (Pierce Company, Minneapolis, MN, USA).
10
Histological Analysis of Lung Tissue in Irradiated Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Left-lung tissues of irradiated mice were fixed in 4% paraformaldehyde and then dehydrated and embedded in paraffin. For histological study, 4 μm tissue sections were stained with haematoxylin and eosin (H&E), Massons trichrome (MT) and immunohistochemical (IHC) stains8 (link). For detection of TGF-β1, tissue sections were incubated with an anti-TGF-β1 primary antibody (1:100 dilution; ab64715, Abcam) at 4 ℃ overnight. Slides were then incubated with avidin18 (link). Slides were then incubated with avidin–biotin peroxidase complex (ABC kit, Vector Laboratories, CA, USA) 4715, Abcam) and were developed using 3,3′-diaminobenzidine tetrachloride (DAB; Zymed Laboratories, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!