Diaminobenzidine dab

After electrophoresis of the proteins by PAGE, under denaturing or non-denaturing conditions, the separated proteins were electrotransferred to a nitrocellulose membrane. The membrane was blocked with an incubation of 2 h with PBS containing 5% skimmed milk at room temperature. Then the membranes were incubated overnight at 4°C with the mouse serum against r0076 diluted 1:500 in PBS containing 2% skimmed milk. The membrane was then washed three times with PBS buffer and incubated with 4 μg of secondary anti-Mouse-F (ab’) 2-xx-biotin (Molecular Probes) for 2 h at 37°C. Then, horse anti-mouse antibody conjugated with avidin-peroxidase (Sigma-Aldrich) was added and incubated for 1 h. The reaction was developed by adding 0.05% diaminobenzidine (DAB, Roche) in 10 ml of H₂O₂. The image was acquired with the help of Gel documentation system (Bio-Rad) and analyzed with Quantity One 4.5.6 software (Bio-Rad).

Histological sections with 3 µm thick were mounted on glass slides previously salinized and subjected to immunohistochemistry reaction using the indirect streptavidin-biotin method. The sections were deparaffinized in xylol and washed in decreasing concentrations of ethyl alcohol (100%, 95%, 90%, 80% and 70%). Antigenic recovery was performed by immersing the sections in a citrate solution, heated for 20 min in a microwave. The marking of the Ki-67 antigen was performed by incubation with the rabbit anti-mouse monoclonal antibody MIB-1 (Dako, Glostrup, Denmark, at 1:50 dilution), for 30 min. The reaction was revealed using diaminobenzidine (DAB, Ventana Medical Systems, Tucson, AZ, USA) and stained against Meyer’s Hematoxylin. Both steps were performed at an interval of four min each. The positive control was performed with human tonsil and negative control; the primary antibody was replaced by phosphate-buffered saline in the reaction. The positivity of the immunostaining was evidenced by the visualization of a cellular precipitate in a brownish tone. Thus, cells whose nuclei are immunostained (stained in brown), regardless of the intensity of the staining, were considered positive.

Histological sections (3 μm thickness), previously salinized and subjected to the immunohistochemical reaction, were assembled on glass slides using the indirect streptavidin-biotin method. The slides were dewaxed in xylol and washed with lessening concentrations of ethyl alcohol (100%, 95%, 90%, 80%, and 70%). Antigenic recovery was performed by immersing the sections in citrate solution, being heated for 20 min under microwave to potentiate the reaction. Marking of the Ki-67 antigen was performed by incubation with rabbit anti-mouse MIB-1 monoclonal antibody (Dako, Glostrup, Denmark, at 1:50 dilution) for 30 min. The revealing reaction was performed with the use of diaminobenzidine (DAB, Ventana Medical Systems, Tucson, AZ, USA), and counter-colored with Meyer’s Hematoxylin. The two steps were completed in four minutes each. The positive control was performed using human tonsil. For the negative control, the primary antibody in the reaction was substituted with phosphate buffered saline. The positivity of the immunostaining was confirmed from the brownish hued cellular precipitate. Thus, cells with immunolabelled nuclei, being stained in brown, regardless of the labeling intensity, were evaluated as positive. The immunoexpression gradation was established from the percentage of positive cells per 1000 cells counted.

For myofibroblast immunodetection, deparaffinized histological slides 3, 6, and 9 were subjected to endogenous peroxidase activity blockade with 3% hydrogen peroxide and methyl alcohol (10 min in a dark room). Subsequent antigen recovery was performed by moist heat under pressure in 10-mM citrate buffer/pH 6.0 solution. The histological sections were incubated with anti-α-SMA antibody (clone 1A4, Dako, 1:100) for 30 min. The secondary antibody (SABC (streptavidin–biotin complex), catalog number SA1022) was incubated at 37 ℃ for 30 min. The reaction was revealed by incubating the histological slides with diaminobenzidine (DAB, Ventana Medical Systems, Tucson, AZ, USA) in a dark room for 30 min. Counterstaining was performed with Meyer’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). Two histological slides of myofibroma were used as a positive control, whereas the negative control was obtained using two other slides from the same tumor, replacing the primary antibody by TRIS–HCl. The number of α-SMA-positive cells was counted in ten field histological series of each histological Sect. (400 × , analytical area corresponding to 0.025 mm²). Final data were expressed as mean ± standard deviation (SD) cells/field.