Mouse anti fat10 monoclonal antibody

Tissue samples were washed in pre-cooled phosphate-buffered saline three times and lysed in a nondenaturing tissue lysis buffer containing protease inhibitors to extract total proteins. Cell lysate proteins were resolved by polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% normal fetal bovine serum at room temperature for 2 h, incubated with mouse anti-FAT10 monoclonal antibody (dilution, 1:400; Santa Cruz Biotechnology) or anti-β-actin antibody (dilution, 1:200; Zhongshan Golden Bridge, Beijing, China) at 4°C overnight, washed with Tween Tris Base Buffer Solution (commonly known as TTBS) three times, and then incubated with a horseradish-peroxidase-labeled secondary antibody (dilution, 1:400; Zhongshan Golden Bridge) at room temperature for 2 h. After the membranes were washed three times with TTBS, the proteins were detected by enhanced chemiluminescence. Images were obtained using the EC3 Imaging System, and the bands were semiquantitatively analyzed using ImageJ software to calculate the relative expression of FAT10 to β-actin. The experiment was repeated at least three times, with mean values calculated for further analysis.

Tissue samples were fixed in neutral buffered formalin solution, embedded in paraffin, and cut into 4-μm sections. The sections were dewaxed in xylene, hydrated in a graded ethanol series, and subjected to immunohistochemical staining for FAT10 using the streptavidin–peroxidase method. Mouse anti-FAT10 monoclonal antibody (dilution, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, United States) was used as a primary antibody. Following diaminobenzidine staining, yellowish-brown granules present in the nucleus were regarded as positive signals. Five high-power (400×) fields with the most strongly positive signal were selected to count the number of positive cells among 200 tumor cells. The percentage of immunoreactive cells was scored as follows: no staining, 0; 1%-10% staining, 1; 11%-50%, 2; 51%-80%, 3; and 81%-100%, 4. Staining intensity was scored on the following 0-3 scale: negative, 0; light yellowish-brown, 1; yellowish-brown, 2; and brown, 3. Immunoreactive score (IS; 0-7) was calculated as the sum of the score of the percentage of immunoreactive cells and the score of staining intensity. Samples with IS < 4 were considered negative, while those with IS ≥ 4 were considered positive.