K0679

To assess CD8+ infiltration in mammary tumors, IHC assays were performed. Five micrometer paraffin sections, cut transversely, were deparaffinized and rehydrated from xylene through a graded series of ethanol. Antigen retrieval was performed in a high-pH Target Retrieval Solution (pH 9, DAKO S2368) in a microwave for 15 min, followed by 20 min of cooling at room temperature. Then, endogenous peroxidases were blocked with 3% hydrogen peroxide in water (DAKO, K0679) and non-specific staining was blocked with protein block serum-free (DAKO, X0909). Immunostaining with CD8 (Abcam, ab33786, 1:100) was performed overnight following the manufacturer’s protocol for LSAB-HRP immunohistochemistry kit (DAKO, K0679). Slides were examined under a bright-field microscope (Olympus BX 61 with Qiacam scanning camera) and image software package CellSens (Waltham). Micrographs were taken at 20× magnification. Total number of positive cells per field was calculated.

The immunohistochemical analyses were performed on the prostatic tissue from the ventral lobe of the rat prostate. Slides were prepared from 5-µm sections of the formalin-fixed, paraffin-embedded tissues, subjected to antigen retrieval with Tris-EDTA buffer (Proliferating Cell Nuclear Antigen- PCNA) and incubated with trypsin for 15 minutes at 37°C (Alpha Smooth Muscle Actin). Endogenous peroxidase activity was blocked by incubating the slides with 3% H₂O₂ in methanol for 15 minutes followed by applying a protein block (phosphate-buffered saline/bovine serum albumin- 5%). Mouse polyclonal primary antibodies to PCNA (1∶100; Invitrogen, 13-3900) and Alpha Smooth Muscle Actin (Invitrogen, 08-0106) were added and incubated overnight. Next, sections were treated with a biotinylated secondary antibody (K0679; Universal DakoCytomation LSAB Kit, Peroxidase, Glostrup, Denmark) and amplified with a biotin–streptavidin system (K0679; Universal DakoCytomation LSAB + Kit, Peroxidase, Glostrup, Denmark). 3, 3 diaminobenzidine tetrachloride (K3466, DakoCytomation, Glostrup, Denmark) was used as the chromogen. After incubation, the sections were counterstained with Mayer hematoxylin.

Immunohistochemistry (IHC) staining was carried out on paraffin‐embedded liver sections (3 μm) using antibodies against F4/80 (123101, BioLegend) and against Ki67 (550609, BD Pharmingen) separately, and this was followed by counterstaining with hematoxylin. Briefly, sections were deparaffinized and rehydrated, and they then underwent antigen‐retrieved using target retrieval solution (S1699, Dako). Next, the liver sections were treated with 3% H₂O₂ in PBS, which was followed by blocking with 5% bovine serum albumin solution. After incubation with each primary antibody in antibody diluent (ab64211, Abcam), the signals were detected by the Labeled Streptavidin–Biotin (LSAB) staining method (K0679, Dako).