Autostainer link 48 device

Freshly cut TMA sections were immunostained on one day and in one experiment. The mouse monoclonal PSA antibody (Dianova DIA-PSA, clone HAM18) was applied at 1:100 and 1:800. Slides were deparaffinized and exposed to heat-induced antigen retrieval for 15 minutes at 98°C in pH9.0 target retrieval solution (Agilent, Santa Clara, CA, USA) in a PT Link pre-treatment module (Agilent) and stained in an Autostainer Link 48 device (Agilent). Protocol steps include 5 min peroxidase blocking (Agilent REAL), 20 min of primary antibody incubation at room temperature and visualization of the bound antibody using the EnVision Flex Kit (Agilent) according to the manufacturer’s directions. Staining was typically homogenous in the analyzed tissue samples and staining intensity of all cases was semiquantitatively assessed in four categories: negative, weak, moderate, and strong.

Freshly cut 4 µm tissue sections were stained for CD3⁺ T lymphocytes and the subset of CD8⁺ cytotoxic T-cells. Following deparaffinzation, slides were exposed to heat-induced antigen retrieval for at 98 °C in pH 9 target retrieval solution (Agilent, Santa Clara, CA, USA) in a PT Link pre-treatment module (Agilent) and stained in an Autostainer Link 48 device (Agilent) using primary antibodies against CD3 (Dako, rabbit polyclonal antibody, Santa Clara, CA, USA; #IR503; undiluted) and CD8 (DAKO, mouse monoclonal antibody, #IR623, undiluted). Protocol steps were performed according to the manufacturer′s directions and include 5 min peroxidase blocking (Agilent REAL) and 20 min of primary antibody incubation at room temperature followed by visualization of the bound antibody using the EnVision Flex Kit (Agilent).