Anti-Podoplanin/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.

1. Cut and mount 3-5 micron formalin-fixed paraffin-embedded tissues on positive charged slides such as Hydrophilic Plus 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 or EDTA.
5. Any of three heating methods may be used:
a. TintoRetriever Pressure Cooker or Equivalent
Place tissues/slides in a staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA, and place in the pressure cooker. Add 1-2 inches of distilled water to the pressure cooker and turn heat to high. Incubate for 15 minutes. Open and immediately transfer slides to room temperature.
b. TintoRetriever PT Module or Water Bath Method Place tissues/slides in a pre-warmed staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA at 95°-99°C. Incubate for 30-60 minutes.
c. 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. For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
8. Wash slides with ImmunoDNA washer or DI water.
9. Continue IHC staining protocol.