Immpress peroxidase secondary and dab substrate kits

Immunohistochemistry was performed on tumors from 4 mice per treatment condition to quantify FoxP3⁺ tumor infiltrate. Tumors were harvested on day 6 after delivery of 12 Gy or sham RT to the primary tumor in mice bearing a primary or a primary and untreated secondary B78 melanoma tumor. Fresh tumor samples were dissected, cryo-embedded in OCT solution, and sectioned. Frozen sections were fixed in -20°C acetone for 20 minutes, blocked in 5% normal rabbit serum, and labeled overnight at 4°C using a 1:1000 dilution of anti-FoxP3 (clone FJK-16s, eBioscience), anti-CD8 (clone 53-6.7, eBioscience), or non-specific isotype control antibody in 5% rabbit serum PBS with 0.01% Triton x-100. FoxP3 is used as a lineage marker for Tregs, recognizing that this population is functionally heterogeneous and may include rare subsets of other cell types (37 (link)). Labeled cells were detected using the ImmPRESS™ peroxidase secondary and DAB substrate kits from Vector Laboratories. Slides were counterstained with hematoxylin. Three representative images were captured from the cortex of each tumor specimen at 200× magnification using an Olympus BX41 inverted microscope equipped with an Olympus XM10 digital camera. Images were viewed using CellSen Standard software and FoxP3⁺ and CD8⁺ cells were quantified by an individual blinded to the treatment conditions.