Prox 1

6 month-old mouse brains in an AKR background were sectioned and paraffin-embedded [21 (link)]. Sections on slides were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H₂O₂ in methanol for 10 min and non-specific binding with 10% dried milk solution. For this specific experiment, tissue sections were incubated with primary antibodies against human proteins that cross-react with mouse; PROX-1 (1:400; Acris), or VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H₂O₂ [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.

Formalin-fixed, paraffin-embedded tissue Sections (8 µm) from the frontal and occipital cortices were cut from cases 1–6 listed in Table 1. Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H₂O₂ in methanol for 10 min and non-specific binding with 10% dried milk solution. Tissue sections were incubated with primary antibodies; MRC1 (1:1000; R&D systems), PROX1 (1:400; Acris), LYVE1 (1:50; Abcam), LYVE1(1:200; R&D Systems), PDPN (1:350; Sigma-Aldrich), and VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H₂O₂ [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.