SMSCs were seeded in 24-well plates and cultured in DM and transfected. After 72 h, cells were fixed in 4% formaldehyde. Subsequently, the cells were permeabilized by adding 0.1% Triton X-100 for 20 min and blocked with 5% goat serum (Beyotime) for 30 min. After incubation with anti-Myosin heavy chain (MyHC; Santa Cruz; 1:250) at 4 °C overnight, the cells were then incubated with the Rhodamine (TRITC) AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG; ZenBio; 1:1000) at 37 °C for 1 h. The cell nuclei were stained with 4’,6-diamidino-2-phenylindole( DAPI; Beyotime; 1:50) for 5 min. Three images were obtained at random using a fluorescence microscope (Olympus, Japan). The area of myotubes was measured by Image-Pro Plus software.
Affinipure goat anti mouse immunoglobulin g igg
Manufactured by ZenBio
Sourced in China
AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG) is a laboratory reagent used to detect and quantify mouse immunoglobulin G in various immunological assays. It is a purified polyclonal antibody preparation raised in goats against mouse IgG.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Goat serum, by Beyotime (3 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Primary antibody myhc, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Coolaber (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Anti fluorescence quencher, by Beyotime (1 mentions)
4 protocols using affinipure goat anti mouse immunoglobulin g igg
1
Myogenic Differentiation Assay Protocol
2
Immunofluorescence Analysis of Myoblast Differentiation
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We further performed immunofluorescence analysis. The chicken myoblasts were seeded in 24-well plates and transfected at the stage of 90% confluence. After 72 h of transfection, myoblasts were fixed with 4% paraformaldehyde (Beyotime) for 30 min and washed 3 times with PBS. Subsequently, cells were permeabilized for 20 min using 0.1% Triton X-100 (Coolaber, Beijing, China) and blocked for 30 min with goat serum (Beyotime). The cells were incubated at 4℃ overnight with diluted primary antibody-MyHC (Santa Cruz Biotechnology, Dallas, TX, 1:300, SC-32732). After the overnight incubation, the cells were washed three times with PBS. Then, Rhodamine (TRITC) AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG ) (ZenBio, Chengdu, China, 1: 1,000, 511102) was added to the plate and further incubated at 37℃ for 1 h. Thereafter, 4’, 6-diamidino-2-phenylindole (DAPI, Beyotime, 1:50) was added to stain the cell nuclei. Finally, the images were randomly captured with a fluorescence microscope (Olympus, Japan). The area of the stained myotubes was calculated by Image-Pro Plus 6.0 software. Each treatment group has three independent replicates.
3
Immunofluorescence Staining of SMSCs
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After 72 h of transfection, the DM of SMSCs was removed, and the cells were washed with PBS (Gibco) 3 times before SMSCs were fixed with 4% paraformaldehyde for 30 min. Subsequently, 0.1% Triton X-100 was used to permeate the SMSCs. The 5% goat serum (Beyotime) was used to block the SMSCs so that the cells could better bind to the primary antibody of Myosin (Santa Cruz, United States; 1: 200). SMSCs were incubated with Rhodamine (TRITC) AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG) (ZenBio; 1: 1000) at 37°C for 1 h. Then, DAPI (Beyotime; 1: 50) was used to stain the cell nuclei before the required images were captured. The results were determined using Image-Pro Plus software.
4
Immunofluorescence Assay of Myotubes
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SMSCs were seeded in 12-well plates and cultured in DM and transfected. After the cells grew and differentiated into myotubes, they were fixed with 4% formaldehyde for 20 min and washed with PBS for 3 consecutive times for 5 min. Subsequently, 0.1% Triton X-100 was added and cultured for 20 min to promote cell permeability, followed by the addition of 5% goat serum (Beyotime, Shanghai, China) for 30 min to block the cells. Furthermore, the primary antibody (MyHC; Santa Cruz; 1:250) was added and incubated overnight at 4 ◦C. After that Rhodamine (TRITC) AffiniPure Goat Anti-Mouse Immunoglobulin G (IgG; ZenBio; 1:1000) was added and the cells were incubated at room temperature for 1 h. Then the cells were rinsed with PBST for three times, thereafter, 4′, 6-diamidino-2-phenylindole (DAPI; Beyotime; 1:50) was added for nuclear staining. Five minutes after staining, the anti-fluorescence quencher (Beyotime) was added and observed under a fluorescence microscope (Olympus, Tokyo, Japan). The mean number of nuclei in each myotubes is equal to the total number of nuclei of the myotubes divided by the total number of myotubes. Three random images were taken from each well and the area, diameter, and average nuclei number of the myotube were measured using Image-Pro Plus software.
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