Vector chromagen dab hrp substrate

Tumors were excised and placed into 10% neutral-buffered formalin saline. Fixed tumors were embedded into paraffin and 5 µm sections were cut from wild-type and mice which carry a point mutation in the ATP-binding site of PI3Kδ which renders this kinase inactive (δ^D910A mice25 ) and from vehicle-treated and PI-3065-treated mice. Sections were stained for high endothelial venules (HEV) as described previously.26 (link) Briefly, antigen retrieval was performed in Tris-EDTA (10 mmol/L; 1 mmol/L, pH 9.0). Endogenous peroxidase activity was quenched using 1% H₂O₂/MeOH and non-specific binding was blocked with 2.5% horse serum. Tumor sections were incubated overnight at 4°C with rat peripheral node addressin (PNAd) (clone MECA79, Biolegend). Sections were then washed and incubated in anti-Rat ImmPRESS HRP polymer detection reagent (VectorLabs). Slides were incubated briefly in Vector Chromagen DAB HRP substrate (Vector Laboratories). Slides were then rinsed with dH₂O and then counterstained with hematoxylin and mounted in DPX. Sections were imaged at 40× magnification using a Zeiss Axio Scan.Z1 slide scanner (Plan-Apochromat 40×/0.95 Korr M27). The total HEV area within each tumor was then calculated using Zen software.2 27 (link)