AutoRED AP Plus Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. AutoRED AP Plus 1X Working Solution: In a clean dark bottle, mix equal volumes of AutoRED AP Plus Buffer and AutoRED AP Plus Chromogen in a 1:1 ratio.
2. MAKE FRESH: WORKING SOLUTION IS STABLE FOR ONLY 20-30 MINUTES.
3. There is no preparation required for automation use. Reagents can be loaded directly on the instrument for on-slide mixing or on-board mixing.

Staining Procedure
1. Once sections have been incubated with alkaline phosphatase, wash sections with wash buffer.

a. On-Slide Mixing (Automation): AutoRED AP Plus has been formulated for direct application of the component solutions onto the slide in a specified order: sequential application of substrate buffer first, followed by chromogen in equal volumes with no washing or reagent "blow off" in between. Reversing the order of reagent application will increase background and yield suboptimal results. Incubate substrate buffer for 1-5 minutes. Incubate chromogen for 10-20 minutes.
b. Batch Mode (Automation): Using Batch Mode on your instrument, wait for machine to notify you when ready, then mix chromogen and substrate-buffer in a 1:1 ratio and load onto instrument. For the best results, look under the microscope for the best signal development. Incubate working solution for 10 - 20 minutes.
c. On Board Mixing (Automation): Instruments that have on-board mixing capability can load the chromogen and substrate-buffer components independently. Working solution is made mixing reagents 1:1 in on-board mixing station before application to slide. Incubate working solution for 10 - 20 minutes.
d. Manual Use: Mix chromogen and substrate-buffer in a 1:1 ratio and apply directly to slide. Working solution is stable for only 20-30 minutes and should be applied immediately for best results. Incubate working solution for 10 - 20 minutes.

2. Counterstain with Hematoxylin for good contrast. Wash with DI H2O followed by immuno wash buffer.
3. Sections can be dehydrated in alcohol, cleared in xylene or xylene substitute and permanently mounted.
4. Alternatively, slides can be air dried (instead of alcohol and xylene). After rinsing off counterstain in DI H2O, leave slides on benchtop for at least 20 minutes to air dry, then permanently mount or use aqueous mounting media.
Recommendation: For best color preservation and long term slide storage, we recommend using Tissue Preservation Solution - HRP/AP assays after counterstaining.