Podoplanin/D2-40

Podoplanin is a transmembrane mucoprotein (38 kDa) recognized by the D2-40 monoclonal antibody. Podoplanin is specifically expressed in the endothelium of lymphatic capillaries but not in the blood vasculature. In normal skin and kidney, podoplanin is co-localized with VEGFR3/FLT4, another marker for lymphatic endothelial cells.

Podoplanin is selectively expressed in lymphatic endothelium as well as Lymphangiomas, Kaposi’s Sarcomas and in subset Angiosarcomas with probable lymphatic differentiation. Podoplanin has also been shown to be expressed in Epithelioid Mesotheliomas, Hemangioblastomas and Seminomas.

Anti-D2-40 is a mouse monoclonal antibody derived from cell culture supernatant that is concentrated, dialyzed, filter sterilized and diluted in buffer pH 7.5, containing BSA and sodium azide as a preservative.

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1. Cut and mount 3-4 micron formalin-fixed paraffin-embedded tissues on positive charged slides.
2. Air dry for 2 hours at 58° C.
3. Deparaffinize, dehydrate and rehydrate tissues.
4. Subject tissues to heat epitope retrieval using a suitable retrieval solution such as ImmunoDNA Retriever with Citrate (BSB 0020-BSB 0023) or EDTA (BSB 0030-BSB 0033).
5. Any of three heating methods may be used:
   • Electric Pressure Cooker
     Place standoff rack at base of pressure cooker. Add 1-2 inches of distilled water to the pressure cooker and turn heat to high, and incubate for 15 minutes. Open and immediately transfer slides to room temperature.
   • Water Bath Method
     Place tissues/slides in a pre-warmed staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA in a water bath set at 95°-99° C. Incubate for 30-60 minutes.
   • Conventional Steamer Method
     Place tissues/slides in a pre-warmed staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA in a Steamer, cover and steam for 30-60 minutes.
6. After heat treatment, transfer slides in ImmunoDNA Retriever with Citrate or EDTA to room temperature and let stand for 15-20 minutes.
7. Wash slides with IHC wash buffer or DI water.
8. Continue IHC staining protocol.