Ab25101

Immunohistochemical analysis was performed as described previously [33 (link)]. Antigen retrieval was performed by soaking the specimen on slides in 5 M sodium citrate solution in phosphate-buffered saline containing 0.05% (v/v) Tween 20 (PBST) (pH 6.0). The slides were subsequently blocked with serum-free protein block (Dako, 2016–08) for 30 min at room temperature and incubated at 4 °C overnight optimally with rabbit polyclonal antibodies to IFN-γ (ab25101, Abcam Inc., Boston, MA, USA) or IL-6 (21865–1-AP, Proteintech Inc., Rosemont, IL, USA) diluted 1:200 in DAKO antibody diluent. After washing three times with PBST, the slides were incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase-labeled polymer (DakoCytomation, Glostrup, Denmark) and subsequently visualized by treatment with 3,3′ diaminobenzidine (DAB) Chromogen (DakoCytomation, #K3465) according to the instructions provided by the manufacturer. Nuclei were visualized using Mayer’s hematoxylin (MERCK, 1:1000 dilution in PBST). For mounting, the sections were rinsed with water, dehydrated in graded ethanol (90% ethanol for 30 s × 3 and 100% ethanol for 30 s × 3), cleared in xylene (for 30 s × 2), and sealed using multi-mount 480 (Matsunami, FM48001). Images were acquired and processed digitally.