Immunofluorescence blocking buffer (5% normal serum) recipe
Blocks non-specific binding in IF and IHC: PBS + 5% normal serum + 0.3% Triton X-100. Also used to dilute antibodies.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Normal serum (from the secondary-antibody host) | 500 µL | 5% |
| 10% Triton X-100 | 300 µL | 0.3% |
| 1X PBS | to final volume |
Steps
- Mix the serum and Triton into PBS.
- Use the same day or keep at 4 °C for 1–2 days.
- Block for 1 h at room temperature (go straight to primary antibody without washing).
Good to know
- Match the serum to the secondary-antibody host (goat anti-rabbit → normal goat serum).
- 1–3% BSA can replace serum.
FAQ
What if I use serum from the primary-antibody species?
The secondary will bind the serum IgG and raise background badly — avoid it.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.