After cells climbing slides, the sample was fixed with 4% cold paraformaldehyde for 30 min and 0.1% Triton‐100 for 10 min to break the cell membrane. Then, 5% goat serum solution was added to block for 30 min. Immunofluorescence staining was performed with anti‐Pax7 (20570‐1‐AP; Proteintech, Wuhan, China) and MyoD (ab16148; Abcam, Cambridge, UK) antibodies. Secondary antibodies, anti‐rat (CW0102S; CWBIO, Taizhou, China) and anti‐rabbit (CW0103S; CWBIO), were added at 37 °C for 1 h. The sample was sealed with an anti‐fluorescence decay seal containing 4′,6‐diamidino‐2‐phenylindole (Solarbio) and observed under fluorescence microscope (Nikon, Tokyo, Japan).
Ab16148
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab16148 is a monoclonal antibody that recognizes the KLF2 protein. KLF2 is a transcription factor involved in regulating gene expression. This antibody can be used for various applications, including Western blotting, immunoprecipitation, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Ab124800, by Abcam (3 mentions)
Ab1835, by Abcam (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Ab125078, by Abcam (2 mentions)
Ab187339, by Abcam (2 mentions)
Ab199010, by Abcam (2 mentions)
Wl02785, by Wanlei (2 mentions)
Wl02028, by Wanlei (2 mentions)
Wl03069, by Wanlei (2 mentions)
Km9001t, by Sungene Biotech (2 mentions)
Goat anti rabbit igg h l hrp, by Boster Bio (2 mentions)
Wl01435a, by Wanlei (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Ab8245, by Abcam (2 mentions)
Fluorescence microscope, by Solarbio (1 mentions)
Cw0102, by CWBIO (1 mentions)
Cw0103, by CWBIO (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Ab34360, by Abcam (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
16 protocols using ab16148
1
Immunofluorescence Staining of Pax7 and MyoD
2
Myo-D and Pax-7 Immunolabeling Protocol
3
Quantifying Myogenic Regulatory Factors in Skeletal Muscle
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Frozen gastrocnemius was crushed using a mortar and a pestle cooled with liquid nitrogen. Then, 50 mg of pulverized tissue was homogenized in a buffer containing 15 mM HEPES, 10% glycerol, 0.5% NP-40, 250 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride (PMSF), and a phosphatase- and protease-inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined by the BCA. Then, 40 μg of protein was subjected to 10–15% SDS-PAGE and transferred to nitrocellulose membranes. After blocking in 3% skimmed milk (1 h, RT), membranes were incubated overnight at 4 °C with anti-myostatin/GDF8 antibody (0.4 μg/mL; R&D Systems, AF788, Minneapolis, MN, USA), pSTAT3 (2.5 μg/mL; R&D Systems, MAB4607), pSTAT1 (5 μg/mL; Affymetrix eBioscience, 14-9008, San Diego, CA, USA), SOCS1 (1 μg/mL; Abcam, ab9870, Cambridge, UK), SOCS3 (4 μg/mL; Abcam, ab14939), Pax7 (2.8 μg/mL, Developmental Studies Hybridoma Bank, AB 528428, Iowa City, IA, USA), Myogenin (2 μg/mL; Abcam, ab82843), and MyoD1 (1 μg/mL; Abcam, ab16148). Antibody binding was detected with chemiluminescence using secondary antibodies: anti-goat-HRP 1:5000 (Merck Millipore), anti-rat-HRP 1:20,000 (Fisher Scientific) or anti-mouse-HRP 1:5000 (GE Healthcare LifeSciences, Piscataway, NJ, USA. α-tubulin protein levels were employed as the loading control.
4
Western Blot Analysis of RMS Cell Lines
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Total cell lysates from human RMS cell lines were obtained following lysis in 2%SDS lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, Dallas, Texas). Samples were boiled, vortexed and homogenized through a 28G syringe. 20–40 μg of protein was loaded in 4–20% Mini-Protean TGX gels (Biorad, Hercules, CA) and transferred onto PVDF membranes. Western blot analysis used primary antibodies: rabbit a-MYF5 (1:5000, Abcam ab125078, Cambridge, MA), mouse a-MYOD1 (1:1000, Abcam ab16148, Danvers, MA), rabbit a-MYOD1 (1:1000, Abcam ab133627), rabbit a-GAPDH (1:2000, Cell Signaling 2118), mouse a-TUBULIN (1:2500, Abcam ab4074) and secondary antibodies: HRP anti-rabbit (1:2000, Cell Signaling 7074) or HRP anti-mouse (1:3000, GE Healthcare NA93IV, Marlborough, MA). Blocking was completed using 5% skim milk/TBST. Membranes were developed using an ECL reagent (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA or sensitive SuperSignal West phemto Maximum Sensitivity Substrate, Thermo Scientific, Waltham, MA).
5
Bovine Primary Myoblast Differentiation
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Bovine primary myoblasts were differentiated for 4 days and then were washed three times with PBS buffer (pH 7.4), followed by fixation with 4% paraformaldehyde for 30 min. Then, cells were permeabilized for 15 min in PBS containing 0.5% Triton X-100. Cells were stained overnight at 4°C using MyoD1 antibody (1;500; ab16148; Abcam, Cambridge, UK) and MyHC antibody (1:250; GTX20015; GeneTex, USA), diluted in 1% BSA. We then incubated them at room temperature for 2 h with the corresponding secondary antibody goat anti-mouse IgG (heavy chain and light chain [H&L])-Alexa Fluor 594 (1:500; RS3608; Immunoway, USA), diluted in 1% BSA in PBS. Nuclei were visualized by DAPI stain. Finally, we washed them three times with PBS and observed under a fluorescence microscope (DM5000B; Leica, Germany).
6
Characterization of Heterogeneous Ovine Stromal-Derived Cells
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Heterogeneous SF-SCs were isolated from the femurs of a 6–8-week-old sheep fetus (obtained at the local abattoir) by flushing with L-15 medium supplemented with Anti-Anti (Thermo Fisher Scientific). Cells were then expanded in vitro and verified for multipotency according to differentiation methods for chondrogenesis61 (link), osteogenesis62 (link), and myogenesis63 (link). Differentiated and undifferentiated SF-SCs were fixed in 4% buffered formaldehyde and stained with a wide range of primary antibodies (1 h at room temperature) from Abcam (Amsterdam, Netherlands), including α-SMA (ab32575; 1:500), vimentin (ab8798; 1:100), CD166 (ab235957; 1:200), Ki67 (ab15580; 1:300), estrogen receptor-α (ER-α; ab66102; 1:100), ER-β (ab187291; 1:100), progesterone receptor (PR; ab2765; 1:100), cytokeratin (ab9377; 1:1000), MyoD1 (ab16148, 1:100), RANK (ab13918, 1:100), and DMP1 (ab103203, 1:100). Each primary antibody was conjugated with either CY3 or Alexa Fluor 488 secondary antibodies (Thermo Fisher; 1:300) and DNA was labeled with DAPI. Chondrogenesis was evaluated after alcian blue staining using standard methods.
7
Antibody Validation for Adipogenesis
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The anti-CNOT1 mouse monoclonal antibody was described previously [25 (link)]. Antibodies against GAPDH (#2118), PPARγ (#2443), C/EBPβ (#3087), and Perilipin (#9349) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against C/EBPα (sc-61) was from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against UCP1 (ab10983), MyoD1 (ab16148), and Myogenin (ab124800) were from Abcam (Cambridge, UK). The antibody against Myosin (M4276) was from Sigma Aldrich (St. Louis, MO, USA).
8
Immunofluorescence Staining of Myoblasts and Myotubes
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Cultured myoblasts and myotubes were washed briefly with PBS and fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature, and then permeabilized with PBS containing 0.2% Triton X-100 (Solarbio, Beijing, China) for 10 min. The cells were subsequently washed with PBS 3 times. The cells were blocked with 10% donkey serum, 1% BSA and 0.3 M glycine in PBS at room temperature for 30 min. The primary antibodies were diluted to different concentrations in blocking buffer according to the protocols, and then, the cells were incubated overnight at 4°C. After washing three times with PBS, the cells were incubated with fluorescent dye-conjugated secondary antibodies diluted in blocking buffer for 1 h at room temperature, and this step was performed in the dark. The cells were washed 3 times with PBS, stained with 0.1% DAPI (Sigma-Aldrich, USA) for 15 min and then visualized under a fluorescence microscope (Olympus IX71, Japan). The primary antibodies used were anti-PAX7 (1:200, ab187339, Abcam) and anti-MyoD1 (1:200, ab16148, Abcam). The secondary antibodies used were Alexa Fluor 555-conjugated donkey anti-rabbit IgG (1:1,000, ab150074, Abcam) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1,000, ab150113, Abcam).
9
Optimizing Myogenesis through siRNA Screening
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Gene-specific smart-pool or control siRNAs (Dharmacon, GE Life Sciences, Marlborough, MA) (0.01 μM) were reverse-transfected into cells using RNAiMax lipofectamine transfection reagent (Life Technologies, Waltham, MA) in flat clear bottom 96 well plates. Cells were then fixed at 72 hr post transfection in 4% PFA/PBS, washed in x1 PBS and permeabilized in 0.5% TritonX-100/PBS. Antibodies used were rabbit a-Myf5 (1:400, Abcam ab125078) and mouse a-MyoD (1:200, Abcam ab16148) in 2% goat serum/PBS, Alexa 488 goat anti-mouse (1:1000, Invitrogen A11029) and Alexa 594 goat anti-rabbit (1:1000 Invitrogen A11037). Cells were incubated with DAPI (1 μg/ml), and imaged at 200x using a LSM710 Zeiss Laser scanning confocal microscope. Images were processed in ImageJ and Adobe Photoshop.
For EdU and AnnexinV assays, gene-specific smart-pool or control siRNAs (Dharmacon, GE Life Sciences) (5 μM) were added to Rh18, RD, 381T, RMS559 and Rh3 cells in a 6-well plate and incubated for 48–96 hr prior to analysis.
For EdU and AnnexinV assays, gene-specific smart-pool or control siRNAs (Dharmacon, GE Life Sciences) (5 μM) were added to Rh18, RD, 381T, RMS559 and Rh3 cells in a 6-well plate and incubated for 48–96 hr prior to analysis.
10
Characterization of Muscle Cell Differentiation
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cDNA was synthesized in accordance with the instructions provided in the reverse transcription kit (Vazyme, Shanghai, China). SYBR, diethyl pyrocarbonate water, cDNA, β‐actin as the reference gene, and upstream and downstream primers were mixed proportionally to 10 μL for the polymerase chain reaction. The primer sequences are shown in Table S3 .
After the concentration was determined by the BCA method, SDS/PAGE electrophoresis was performed and the target protein was transferred to the nitrocellulose membrane. The membrane was incubated with primary antibody overnight at 4 °C, followed by secondary antibody for 1 h at 37 °C. The protein was visualized with a gel imager (Bio‐Rad, Hercules, CA, USA). The primary antibodies included Pax7 and anti‐MyoD (ab199010 and ab16148; Abcam); PPARγ and Glyceraldehyde 3‐phosphate dehydrogenase (16643‐1‐AP and 60004‐1‐AP; Proteintech); α‐smooth muscle actin (α‐SMA; 19245, Cell Signaling, Danvers, MA, USA); Collagen‐1 (ab255809; Abcam); anti‐β‐actin (CW0096M; CWBIO), and TSG101 and CD9 (28283‐1‐AP and 20597‐1‐AP; Proteintech).
After the concentration was determined by the BCA method, SDS/PAGE electrophoresis was performed and the target protein was transferred to the nitrocellulose membrane. The membrane was incubated with primary antibody overnight at 4 °C, followed by secondary antibody for 1 h at 37 °C. The protein was visualized with a gel imager (Bio‐Rad, Hercules, CA, USA). The primary antibodies included Pax7 and anti‐MyoD (ab199010 and ab16148; Abcam); PPARγ and Glyceraldehyde 3‐phosphate dehydrogenase (16643‐1‐AP and 60004‐1‐AP; Proteintech); α‐smooth muscle actin (α‐SMA; 19245, Cell Signaling, Danvers, MA, USA); Collagen‐1 (ab255809; Abcam); anti‐β‐actin (CW0096M; CWBIO), and TSG101 and CD9 (28283‐1‐AP and 20597‐1‐AP; Proteintech).
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