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Rapid Staining Procedure for VECTASTAIN Elite

This method is an adaption of the recomended protocol when using the VECTASTAIN® Elite ABC kits. The increased concentration of reagents allows for decreased incubation times.

The sensitivity of the VECTASTAIN® Elite ABC Kit permits development of shortened immunoperoxidase staining protocols. These guidelines are provided for a rapid staining method having a sensitivity and staining quality equivalent to the full-length VECTASTAIN® Elite ABC protocol.


  1. Prepare paraffin-embedded or frozen sections for staining as described elsewhere. Prepare VECTASTAIN® Elite ABC Kit reagents as follows:
    • For the Biotinylated Antibody, add one drop concentrated stock to 5 ml of PBS containing 1.5% normal serum. If background staining is a problem, increase the concentration of normal serum up to 10%.
    • For the VECTASTAIN® Elite ABC Reagent, add two drops of Reagent A to 2.5 ml buffer, mix, then add two drops of Reagent B. Mix and allow to stand for 5-30 minutes before use.
  2. If quenching of endogenous peroxidase is required, an accelerated quenching procedure can be employed. Treat sections with 3% hydrogen peroxide in water for 3-5 minutes.
  3. Wash gently with a stream of buffer from a wash bottle.
  4. If background staining is a problem, incubate sections for 5-10 minutes in 2% -10% normal serum in buffer.
  5. Incubate sections with primary antibody.*
  6. Wash as in step 3.
  7. Incubate sections for 10 minutes with diluted biotinylated secondary antibody.
  8. Wash as in step 3.
  9. Incubate sections for 5 minutes with VECTASTAIN® Elite ABC Reagent.
  10. Wash as in step 3.
  11. Incubate sections in peroxidase substrate solution until desired stain intensity develops
  12. Wash as in step 3.
  13. Counterstain, clear and mount.**

*The concentration, staining time and temperature of the primary antibody should be tailored to an investigator's particular requirements. The increased sensitivity of the VECTASTAIN® Elite ABC Kit allows shorter primary antibody incubation times. For example, at primary antibody concentrations optimal for the regular VECTASTAIN® ABC Kit, incubation times can be reduced at least in half when using the VECTASTAIN® Elite ABC Kit. Higher concentrations of primary antibody allow even shorter incubation times.

** The dehydration and clearing protocol can be shortened to 2x1 minute washes in isopropyl alcohol if VectaMount Express Mounting Media is used. (H-5700-60)

NOTE: A very rapid procedure that provides excellent staining results can also be performed. Prepare diluted biotinylated secondary antibody 1 drop/2.5 ml. Prepare VECTASTAIN® Elite ABC Reagent as in the above protocol. Apply diluted VECTASTAIN® Elite ABC   Kit   reagents preheated to 37°C. Incubate sections in each reagent for 2 minutes.

A VECTASTAIN® Universal Quick Kit (Cat. No. PK-8800), based on a pre­ formed streptavidin/peroxidase complex, is also available to perform rapid immunohistochemical staining.