Protein was extracted using radioimmunoprecipitation assay (RIPA) buffer (Epizyme, China) containing cocktail proteinase inhibitors. The protein concentration was quantified with a bicinchoninic acid (BCA) protein assay kit (Epizyme, China). The same amount of protein was run on sodium dodecyl sulfate (SDS)-polyacrylamide gels in the Tris/SDS buffer system and then transferred into polyvinylidene difluoride (PVDF) membranes. The membranes and the primary antibodies c-Fos (Bioss, China) were incubated together overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abbkine, China). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Boster, China) was used as an internal reference antibody. The protein expression was analyzed using an electronic shelf label (ESL) scanner with Omni-ECL Enhanced Pico Light Chemiluminescence Kit (Epizyme Biotech, China), and signal intensities of the protein expression were quantified using NIH Image/J software (National Institutes of Health, Bethesda, MD).
C fos
Manufactured by Bioss Antibodies
Sourced in China
C-Fos is a nuclear protein that serves as a transcription factor. It is a proto-oncogene that regulates gene expression. C-Fos plays a role in cell proliferation, differentiation, and transformation.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Ipsen (2 mentions)
Bca protein assay kit, by Ipsen (2 mentions)
Gapdh antibody, by Boster Bio (1 mentions)
Dab substrate solution, by ZSGB-BIO (1 mentions)
Gapdh, by Bioss Antibodies (1 mentions)
Nfatc1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc eq system, by Bio-Rad (1 mentions)
Matrix metalloproteinase mmp 2, by Bioss Antibodies (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Cyclin d1, by Bioworld Technology (1 mentions)
7 protocols using c fos
1
Western Blot Analysis of c-Fos Protein Expression
2
Immunohistochemical Analysis of c-Fos
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Mice brains were put into 4% of paraformaldehyde for at least 24 h at 4°C. The brains were embedded in paraffin after dehydration and clearance. The paraffin-embedded tissue blocks were cut into a section at 4 μm thickness on a microtome. Sections on glass slides were deparaffinized and rehydrated before immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0) for 20 min for antigen retrieval. The endogenous peroxidase was blocked by incubating in 3% of H2O2 solution for 10 min at room temperature. The primary antibody c-Fos (Bioss, China) was added onto the sections for incubating overnight at 4°C. The biotinylated secondary antibody and streptavidin-HRP were sequentially incubated with the sections. The antibody was stained using 3,3′-diaminobenzidine (DAB) substrate solution (Zsbio, China). The slides were counterstained by hematoxylin. The slides were dehydrated and cleared before mounting coverslips. Sections were visualized under a microscope (Olympus, Japan) 100 and 400 times.
3
Immunohistochemical Analysis of c-Fos Expression in Mouse Brain
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Mice brains were put into 4% paraformaldehyde for at least 24 h at 4°C. The brains were embedded in paraffin after dehydration and clearance. The paraffin-embedded tissue blocks were cut into sections at 4 μm thickness on a microtome. Sections on glass slides were deparaffinized and rehydrated before immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0) for 20 min for antigen retrieval. The endogenous peroxidase was blocked by incubating in 3% H2O2 solution for 10 min at room temperature. The primary antibody c-Fos (Bioss, China) was incubated with the sections over night at 4°C. After washing, the biotinylated secondary antibody and streptavidin-HRP were sequentially incubated with the sections. After washing, the sections were stained using DAB substrate solution (Zsbio, China). The sections were counterstained by hematoxylin. Then, the sections were dehydrated and cleared before mounting coverslips. Finally, sections were visualized under a microscope at × 400 or × 100 (Olympus, Japan).
4
Western Blot Analysis of RANKL-Induced Signaling
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Cells were lysed in cell lysis buffer for western blot and Co-IP (Beyotime, Shanghai, China) and 25 µg of protein from each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring the proteins onto PVDF membranes (0.22 µm; Merck Millipore, Darmstadt, Germany), followed by blocking in 5% skim milk for 2 h, the membranes were incubated with the following primary antibodies overnight at 4 °C: klotho (1:500; Bioss, Beijing, China), NFATc1 (1:1,000; Abcam, Cambridge, UK), c-Fos (1:500; Bioss, Beijing, China), and GAPDH (1:1,000; Bioss, Beijing, China). Then, the membranes were washed 3 times with Tris-buffered saline and tween (TBST) followed by incubation with anti-rabbit secondary antibodies (1:1,000; Bioss, Beijing, China) in a blocking solution for 1 h at room temperature. Blots against GAPDH were set as the loading controls. For the detection of NF-κB signaling pathway phosphorylation, IκB, P65, and their phosphorylated proteins were detected. The medium of RANKL-induced BMMs and RAW 264.7 cells at 1 d was replaced by medium without RANKL for 12 h, then cells were treated with or without RANKL for 10 min. The subsequent steps for western blot were the same as mentioned above. Primary antibodies against IκB (1:500), p-IκB (1:500), P65 (1:1,000), and p-P65 (1:1,000) were all supplied by Abcam (Cambridge, UK).
5
Western Blot Analysis of c-Fos in Mouse Brain
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The retrieved mouse brains were stored at −80°C and the proteins were extracted using the RIPA buffer (Epizyme, China). The concentration of the extracted protein was determined using the BCA protein assay kit (Epizyme, China). The same amounts of protein were run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% non-fat milk for 1 h at room temperature. The membranes and the primary antibodies c-Fos (Bioss, China) were incubated together and overnight at 4°C, and then incubated with an HRR-conjugated secondary antibody (Abbkine, China). The GADPH protein (Boster, China) serves as the internal reference protein. The proteins expression was analyzed using an ESL scanner with Omni-ECL Enhanced Pico Light Chemiluminescence kit (Epizyme Biotech, China), and signal intensities of the protein expression were quantified using Chemidoc EQ system (BioRad, USA).
6
Western Blot Analysis of Cell Proteins
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After transfection for 48 h, SK-RG cells were collected and lysed for total protein extraction. The proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% skimmed milk for 2 h, and primary antibodies, including β-actin (1:1,000; ZSGB-Bio, Beijing, China), c-fos (1:1,000; Bioss, Beijing, China), CDK4, CDK6 (1:1,000; Bioworld Technology, St Louis Park, MN, USA), cyclin D1 (1:500; Bioworlde Technology, St Louis Park, MN, USA), matrix metalloproteinase (MMP)2 (1:1,000; Bioss, Beijing, China), and MMP9 (1:1,000; Boster, Wuhan, China), were added overnight at 4 °C. The membranes were washed with 1 × Tris-buffered saline with Tween solution. Following that, secondary goat anti-rabbit or goat anti-mouse antibodies conjugated with horseradish peroxidase (1:10,000; ZSGB-Bio, Beijing, China) were added for 2 h at room temperature. After washing the membranes three times with 1 × PBS, the bands were detected by electrochemiluminescence and analyzed using ImageJ software (1.48v; NIH, Bethesda, MD, USA).
7
Immunohistochemical Analysis of Nude Mice
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Tissues isolated from different groups of nude mice were fixed with 10% formalin and paraffin-embedded to produce tissue sections. After dewaxing the slices, they were boiled for 8 min in sodium citrate buffer (pH = 6.0). The sections were cooled to 30 °C and incubated in 3% hydrogen peroxide at room temperature for 10 min while being protected from light. The sections were washed with PBS (3 × 5 min). Primary antibodies c-fos (1:100; Bioss, Beijing, China), CDK6, and cyclin D1 (1:50; Bioworld Technology, St Louis Park, MN, USA) were applied overnight at 4 °C. The sections were washed with PBS. The secondary antibody (ZSGB-Bio, Beijing, China) was added and incubated for 30 min at 37 °C. After washing again, the slides were colored with 3,3′-diaminobenzidine (DAB) working solution for 60 s at room temperature, and finally, the slides were re-stained with hematoxylin and sealed.
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