BLUE AP Plus Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. Aliquot 1mL of BLUE AP Plus Substrate Buffer in a mixing bottle.
2. Add one drop (~20ul) of concentrated BLUE AP Plus Chromogen solution.
3. Replace tip, mix, and allow solution to reach room temperature before using.

Note: The BLUE AP Plus chromogen-substrate working solution is light sensitive and should be kept away from light as much as possible. Working solution is stable for up to 6 hours in the dark; any solution not used during this period should be discarded. For optimal staining, use freshly made solution.

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

a) Pre-Mix Working Solution: (Automation) BLUE AP Plus working solution has 6 hour stability and can be loaded directly onto instrument as a single solution. Reduce exposure to light to achieve optimal staining. Working solution is applied directly to slide. Incubate for 10 - 20 min
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 in on-board mixing station before application to slide. Incubate for 10 - 20 min.
c) Manual Use: Mix substrate-chromogen and buffer in a 1:50 ratio and apply directly to slide. Incubate 10 - 20 min.

2. Counterstain with Hematoxylin or Nuclear Fast Red for good contrast. Wash with DI H2O followed by immuno wash buffer.
3. We recommend air drying slides (instead of dehydrating or clearing in alcohol and xylene-substitute). After rinsing off counterstain in DI H2O, leave slides on benchtop for at least 20 minutes to air dry, and then permanently mount.
4. Alternatively, you may dehydrate sections in increasing concentrations of ethanol up to 100%, clear in a xylenesubstitute*, and mount with a permanent mounting medium.
*Notes: Use xylene-substitute instead of xylene.
Recommendation: For best color preservation and long term slide storage, we recommend to use Tissue Preservation Solution - HRP/AP assays after counterstaining.