AutoRED AP Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. Aliquot 1mL of AutoRED AP Substrate Buffer in a mixing bottle.
2. Add one drop (~20ul) of concentrated AutoRED AP Chromogen solution.
3. Replace tip, mix, and allow solution to reach room temperature before using.
4. The working chromogen-substrate solution should be prepared fresh and used within 20-30 minutes of preparation.
5. Any solution not used during this period should be discarded.

Staining Procedure
1. Once sections have been incubated with alkaline phosphatase wash sections with wash buffer then follow protocol of choice:

a. Batch Mode: (Automation) Using Batch Mode on your instrument, wait for machine to notify you when ready, then mix chromogen and buffer in a 1:50 ratio and load onto instrument. Working solution is stable for only 20-30 minutes and should be applied to slide immediately for best results. Incubate for 10 - 20 minutes.
b. On Board Mixing: (Automation) Instruments that have onboard mixing capability can load the chromogen and substrate-buffer components independently. Working solution is made mixing reagents 1:50 using on-board mixing station before application to slide. Incubate for 10 - 20 minutes.
c. Manual Use Mix substrate-chromogen and buffer in a 1:50 ratio and apply directly to slide. Incubate for 10 - 20 minutes.

2. Counterstain with Hematoxylin or other counterstain.
3. Wash with DI H2O followed by immuno wash buffer.
4. Sections can be dehydrated in alcohol, cleared in xylene or xylene substitute and permanently mounted.
Note: 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.