What is Normal Donkey Serum (NDS)?

Why donkey serum became the default for multi-label IF

When multiple primary antibodies from different host species are used simultaneously (e.g. mouse anti-CD3 + rabbit anti-Ki67 + goat anti-CK5), secondary antibodies must each recognise a different primary species without cross-reacting with each other. Donkey-raised secondaries (donkey anti-mouse, donkey anti-rabbit, donkey anti-goat) are available pre-adsorbed against all common species — making donkey the single species covering all primary hosts with one NDS blocking step.

NDS blocking protocol

1. After antigen retrieval, drain excess buffer
2. Apply 5–10% NDS in PBS, 200–300 µL per section
3. Incubate 30–60 min at room temperature
4. Drain (do not wash) — apply primary antibody in 1–5% NDS/PBS

Key Facts

• Species matching rule: blocking serum species must match the species in which the secondary antibody was raised
• If secondary is donkey anti-rabbit AF488 → blocking serum must be Normal Donkey Serum (NDS)
• NDS is the default for multi-label IF because most commercial cross-adsorbed secondaries are donkey-raised
• For multi-label with 3–4 primaries: use single-species donkey secondaries + one NDS blocking step — simplest protocol
• Recommended: 5–10% NDS in PBS, 30–60 minutes at room temperature, do not wash before primary antibody
• Available from SeamlessBio: 50 mL, 100 mL, 500 mL; heat inactivated on request

Further Reading

• Normal Serum for IHC/IF Blocking Guide
• What is Horse Serum?
• Animal Sera Portfolio

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