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.
- 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.
- 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.
- Wash gently with a stream of buffer from a wash bottle.
- If background staining is a problem, incubate sections for 5-10 minutes in 2% -10% normal serum in buffer.
- Incubate sections with primary antibody.*
- Wash as in step 3.
- Incubate sections for 10 minutes with diluted biotinylated secondary antibody.
- Wash as in step 3.
- Incubate sections for 5 minutes with VECTASTAIN® Elite ABC Reagent.
- Wash as in step 3.
- Incubate sections in peroxidase substrate solution until desired stain intensity develops
- Wash as in step 3.
- 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.