Rabbit anti human mtor monoclonal antibody

Fifth generation-cultured fibroblast cells were taken, 4% paraformaldehyde was added, and fibroblast cells were fixed for 30 min at 25°C, and washed three times with PBS buffer liquid. The cells were then incubated for 20 min with 0.1% Triton X-100 at 37°C, washed with PBS liquid, and sealed for 1 h with non-immune goat serum. When the serum was absorbed completely, primary antibody was added. Rabbit anti-human mTOR monoclonal antibody (dilution: 1:500; catalog no. 8665T) and rabbit anti-human 70S6K monoclonal antibody (dilution: 1:500; catalog no. 2903T) were obtained from Cell Signaling Technology (Danvers, MA, USA). The cells were incubated overnight at 4°C, washed with PBS liquid the following day, and FITC-labeled goat anti-rabbit secondary antibody (dilution: 1:200; catalog no. 4412S; Cell Signaling Technology) was added. Then they were incubated for 60 min at 37°C, washed with PBS liquid, redyed with DAPI in the dark, and observed under a fluorescence microscope (Olympus Corp.) after 10 min.

The 5th generation fibroblasts were taken, 4% paraformaldehyde was added, fixed at 25°C for 30 min, washed three times with phosphate buffer solution (PBS), incubated at 0.1% Triton X-100 37°C for 20 min, washed with PBS, and sealed with non-immune goat serum for 1 h. Primary antibody was added after the complete suction of serum, and they were incubated overnight at 4°C. Rabbit anti-human TGF-β1 monoclonal antibody (dilution: 1:500; catalog no. 5154LC) and rabbit anti-human PI3K monoclonal antibody (dilution: 1:500; catalog no. 9655T), rabbit anti-human protein kinase B (Akt) monoclonal antibody (dilution: 1:500; catalog nos. 9474S) and rabbit anti-human mTOR monoclonal antibody (dilution: 1:500; catalog no. 8665T) were from Cell Signaling Technology (Danvers, MA, USA). The following day, after they were washed with PBS, secondary antibody (dilution: 1:200; catalog no. 4412S; Cell Signaling Technology) was added, incubated at 37°C for 60 min, washed with PBS, re-dyed with DAPI in the dark, and observed under fluorescence microscope (Olympus Corp.) after 10 min.