CD3

The CD3 antigen is a protein complex composed of three distinct chains (CD3γ, CD3δ and CD3ε) that associate with T-cell receptors and the ζ-chain to generate an activation signal in T-lymphocytes. The TCR, ζ-chain and CD3 molecules together comprise the TCR complex. The CD3γ, CD3δ and CD3ε chains are highly-related cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif (or ITAM for short), which is essential for the signaling capacity of the TCR. Phosphorylation of the ITAM on CD3 renders the CD3 chain capable of binding the enzyme ZAP70 (zeta-associated protein), a kinase important in the signaling cascade of the T-cell.

CD3 has been considered the best all-around T-cell marker. This antibody reacts with an antigen present in early thymocytes. The positive stainingof this marker may represent a sign of early commitment to the T-cell lineage.

CD3 is a rabbit 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.