VECTASTAIN® ABC-AP KIT - Vector Laboratories

VECTOR
LABORATORIES
IVD
VECTASTAIN ® ABC-AP KIT
Catalog Number AK-5000 Series
INSTRUCTIONS FOR IMMUNOHISTOCHEMICAL STAINING
The VECTASTAIN ® ABC system is widely accepted as one of the most sensitive, economical and reliable detection systems
available. The general procedure for staining with a VECTASTAIN ® ABC system is as follows: unlabeled primary antibody
is applied, followed by biotinylated secondary antibody, and then a preformed Avidin and Biotinylated enzyme
macromolecular Complex. The enzyme is visualized by the development of a substrate that produces a color. This has been
termed the ABC technique.
VECTASTAIN ® ABC-AP KIT CONTENTS
VECTASTAIN ® ABC-AP Kits contain the following:
• 3 ml Blocking Serum (Normal Serum)
• 1 ml Biotinylated Secondary Antibody (1.5 mg for anti-IgG, 0.5 mg for anti-IgM antibodies)
• VECTASTAIN ® ABC-AP Reagent: 2 ml Reagent A (Avidin DH solution) and 2 ml of Reagent B (biotinylated alkaline
phosphatase)
• 3 empty mixing bottles
• Detailed instructions for use
* VECTASTAIN ® ABC-AP Kit (Standard), Cat. No. AK-5000, contains only 2 ml Reagent A (Avidin DH solution) and 2 ml of
Reagent B (biotinylated enzyme)
VECTASTAIN ® ABC-AP Kit should be stored at 2 - 8 °C.
PREPARATION OF VECTASTAIN ® WORKING SOLUTIONS
For convenience, VECTASTAIN ® ABC-AP Kits include mixing bottles to prepare working solutions of reagents. To remove the
drop dispenser tip for refilling, press laterally with thumb until the tip snaps off. When dispensing drops, hold the bottle
upside-down and squeeze gently. Put the caps on the bottles when not in use. Once finished staining, discard remaining
dilute working solutions, wash the containers with distilled water, and store all bottles in the original box.
Apply enough solution on the slide to cover the entire section. Incubate slides in a humidified chamber. Staining dishes,
Coplin jars, or automated tissue staining instruments may also be used in the staining procedure.
A number of different washing buffers can be used in the VECTASTAIN ® ABC-AP system. One of the most common is 10 mM
sodium phosphate, pH 7.5, 0.9% saIine (PBS). Prepare the working solutions as follows:
• Blocking Serum (Normal Serum): add three (3) drops (150 µl) of stock (yellow label) to 10 ml of buffer in mixing
bottle (yellow label). The preferred serum for blocking is from the same species in which the biotinylated secondary
antibody is made.
• Biotinylated Secondary Antibody: add one (1) drop (50 µl) of biotinylated antibody stock to 10 ml buffer in mixing
bottle (blue label).
• VECTASTAIN ® ABC-AP Reagent: add exactly two (2) drops of REAGENT A (red label) to 10 ml of buffer in the ABC
Reagent large mixing bottle. Then add exactly two (2) drops of REAGENT B (red label) to the same mixing bottle. Mix
immediately, and allow VECTASTAIN ® ABC-AP Reagent to stand for about 30 minutes before use.
Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010 U.S.A.
Tel: (650)697-3600 • Fax (650)697-0339 • Email: vector@vectorlabs.com • Website: www.vectorlabs.com

ENZYME SUBSTRATES
A variety of chromogens are used with the VECTASTAIN ® ABC-AP system to visualize alkaline phosphatase in tissue sections.
The following is a list of alkaline phosphatase substrates and the colors they produce:
• Vector ® Red Red
• Vector ® Black Black
• Vector ® Blue Blue
• BCIP/NBT Blue/Purple
* Vector ® Blue is soluble in xylene, hence xylene substitutes must be used to clear the tissue sections and non-xylene
mounting media must be used. All other substrates can be permanently mounted in non-aqueous mounting media.
These substrates can be used to introduce multiple colors in a tissue section.
STAINING PROCEDURE FOR PARAFFIN SECTIONS
1. Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series.
2. Rinse for 5 minutes in tap water.
3. Wash in buffer for 5 minutes.
4. Incubate sections for 20 minutes with diluted normal blocking serum.
5. Blot excess serum from sections.
6. Incubate sections for 30 minutes with primary antibody diluted in buffer.
7. Wash slides for 5 minutes in buffer.
8. Incubate sections for 30 minutes with diluted biotinylated secondary antibody.
9. Wash slides for 5 minutes in buffer.
10. Incubate sections for 30 minutes with VECTASTAIN ® ABC-AP Reagent.
11. Wash slides for 5 minutes in buffer.
12. Incubate sections in alkaline phosphatase substrate solution until desired stain intensity develops.
13. Rinse sections in tap water.
14. Counterstain, clear and mount.
*If antigen unmasking is required, perform this procedure after step 2, using a suitable Antigen Unmasking Solution,
such as Cat. No. H-3300 (Low pH) or Cat. No. H-3301 (High pH).
STAINING PROCEDURE FOR FROZEN SECTIONS
This procedure is generally appropriate for frozen sections, cell smears or cytocentrifuge preparations.
1. Sections are air dried.
2. Immediately before staining, fix sections with acetone or the appropriate fixative for the antigen under study.
3. Transfer slides into buffer.
4. Follow steps 4-14 of the procedure recommended for paraffin sections.
If unwanted staining occurs in the absence of biotinylated secondary antibody, endogenous biotin may be present in the
tissue. To eliminate this unwanted staining, use an Avidin/Biotin blocking step (Cat. No. SP-2001) between steps 5 and 6.
DISPOSAL OF REAGENTS
Flush to sewer if allowed or dispose of reagents according to local regulations.
MSDS available online at: www.vectorlabs.com
For Professional Use Only.
Authorized Representative / Exclusive Distributor to UK & Ireland:
Vector Laboratories Ltd
3 Accent Park
Bakewell Road, Orton Southgate
Peterborough, PE2 6XS, United Kingdom,
Tel: (01733) 237999 • Fax: (01733) 237119
Revised 12/09