Ultravision lpvalue detection system

The cells of IV th and Х th passages were seeded on cover glasses and grew for 48-72 h. After the monolayer reached about 50%-70% confluency, the cells were fixed in fixing solution (methanol + acetone, 1:1) for 2 h at -20ºC, washed several times with PBS, incubated with a 1% solution of bovine serum albumin (BSA) for 20 min, and treated with monoclonal antibodies against: For visualisation of reactions the Ultra Vision LPValue Detection system (Thermo Scientific), which contain detecting antibody, conjugated with peroxidase, was used. Enzyme activity was detected by using of diaminobenzidine (Thermo Scientific) as a substrate (Detre et al., 1995) (link). After conducting an immunocytochemical reaction, the preparations werewashed with water and stained with Mayer haematoxylin (Sigma) for 1-2 min, and placed in Faramount Aqueous Mounting Medium (Gluzman et al., 2000) . The results were analysed by counting the number of positively stained cells (brown staining) and evaluated by theclassical H-Score method: S = 1xA+ 2xB + 3xC, where S -H-Score index. The values range from 0 (antigen not detected) to 300 (strong expression in 100% of cells); A -percentage of weakly stained cells; B -percentage of moderately stained cells; C -percentage of strongly stained cells