Immunohistochemical Analysis of Tumor Infiltrating Lymphocytes

Formalin-fixed paraffin-embedded tumor tissue specimens were prepared as described previously [18 (link)]. For immunohistochemistry (IHC), the tumors were sliced into 4-μm-thick sections, deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01 M PBS, pH 7.4. Heat-induced epitope retrieval was performed using citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA), followed by blocking with a blocking buffer (Dako). The tissue sections were stained with anti-CD4 and CD8 antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies and 3,3′-diaminobenzidine substrate chromogen solution (DAB, Dako). IHC images were obtained using an Aperio ScanScope AT slide scanner (Leica Biosystems Inc., Buffalo Grove, IL, USA) and analyzed using ImageScope software 12.4.3 (Leica Biosystems, Buffalo Grove, IL, USA).