Immunohistology procedure
tumor
TUMOR, PATHOLOGY
Biopsies were collected before study entry (archival materials), at baseline or during treatment and were fixed after collection in paraformaldehyde and embedded in paraffin by the pathology laboratory. Four-micron slices were cut from FFPE tumor samples. The tissues embedded in paraffin were cut on a Leica rotary microtome (RM2145). For CD8 and PD-L1 staining, slides were deparaffinized and stained using a PT link (Agilent) and an Autostainer 48 (Agilent). In brief, slides were deparaffinized using a pH 9 buffer for 25 min at 95 °C. After cooling, slides were washed in wash buffer (Agilent) twice for 5 min. Peroxydase blocking was performed with peroxydase blocking reagent (SM801, Agilent). Then, anti-human CD8 (1:100, clone C8/144B, M7103, Agilent) or anti-human PD-L1 (1/200, clone QR1, C-P0001-01, Diagomics) was added for 30 min at room temperature. EnVision FLEX HRP polymers (SM802, Agilent) were added for 15 min at room temperature after two washing steps. DAB (SM803, Agilent) was then added to samples for 2 min. After two new washing steps, slides were finally incubated with hematoxylin (SM806, Agilent) for 20 min and permanently mounted using a Leica automated coverslipper. For the double staining procedure, after antigen retrieval as previously described, anti-decorin antibody (1:100, clone E2N2C, 85786, CST) was added for 30 min, and, after amplification steps as previously described, HRP Magenta (GV92511-2, Agilent) was added for 5 min. Antibody elution was next performed with stripping solution