Colon sections were prepared as described above, deparaffinized, and rehydrated in descending ethanol series before heat-induced antigen retrieval (20 minutes; Antigen Decloaker, Biocare Medical, Concord, CA), blocked in 5% milk for 2 hours at room temperature (RT), and incubated overnight at 4°C with an rabbit anti-mouse lymphatic vascular endothelial hyaluronan receptor 1 antibody (LYVE-1, 1:150; Abcam, Cambridge, MA). Slides were washed in amplifying wash buffer (Bioworld, Dublin, OH), reacted in alkaline phosphatase conjugated secondary antibody (goat anti-rabbit alkaline phosphatase, 1:50; Sigma) for 1 hour, and washed again in amplifying wash buffer (Bioworld). To visualize LYVE-1 positive staining, a Warp Red chromogen kit (Biocare Medical, Concord, CA) as alkaline phosphatase substrate was used. Slides were counterstained with hematoxylin, washed with Tris-buffered saline (TBS), dried, and dehydrated in ethanol and xylene, and mounted in permanent mounting medium (ThermoFisher Scientific).
Becker F., Kurmaeva E., Gavins F.N., Stevenson E.V., Navratil A.R., Jin L., Tsunoda I., Orr A.W., Alexander J.S, & Ostanin D.V. (2016). A Critical Role for Monocytes/Macrophages During Intestinal Inflammation-associated Lymphangiogenesis. Inflammatory bowel diseases, 22(6), 1326-1345.
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