Tumortacs in situ apoptosis detection kit

Tissue blocks were fixed in 10% formalin. After paraffin embedding, 3-μm sections were subjected to staining. For the cell number counts, 5 randomly selected sites at 400× magnification (high-power field) were evaluated by use of light microscopy. For immunohistochemistry of CD4, CD8, or FoxP3, the sections were deparaffinized in xylene and rehydrated before antigen retrieval by boiling in citrate buffer (0.01 M citrate containing 0.5% Tween 20, pH 6.0). The sections were incubated in 10% bovine serum albumin (BSA) in PBS at room temperature for 1 h and then stained with rat anti-CD4 antibody (4SM95, 1:500 dilution; eBioscience, San Diego, CA, USA), anti-CD8 antibody (4SM15, 1:500 dilution; eBioscience), or anti-FoxP3 antibody (FJK-16s, 1:200 dilution; Invitrogen, Waltham, MA, USA) overnight at 4 °C, followed by biotinylated anti-rat IgG antibody (1:500; Vector Laboratories, Burlingame, CA, USA) and Vectastain ABC reagent (Vector Laboratories) at room temperature for 60 min and 30 min, respectively. Finally, the sections were stained by the use of a DAB Peroxidase Substrate Kit (Vector Laboratories) before imaging. For detection of apoptotic cells, a TumorTACS in Situ Apoptosis Detection Kit (Trevigen, Gaithersburg, MD, USA) was used according to the manufacturer’s instructions.

Paraffin-embedded tumor sections (n = 8/group; Three micrometer thick) were processed with the Tumor TACS In Situ Apoptosis Detection kit (Trevigen)^{9 (link)} or with Click-iT TUNEL Colorimetric ICH Detection kit (Invitrogen) according to manufacturer’s protocols. Apoptotic nuclei labeled by diaminobenzidine were counted on 10 squares/sample (n = 8/group). Magnification 20 × .