GREEN AP Plus Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. Aliquot 1mL of GREEN AP Plus Substrate Buffer in a mixing bottle.
2. Add one drop (~20ul) of concentrated GREEN AP Plus Chromogen solution.
3. Replace tip, mix, and allow solution to reach room temperature before using.
4. GREEN AP Plus chromogen-substrate working solution is light sensitive and should be kept away from light as much as possible.
5. Working solution should be prepared fresh in the dark.

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

a. Pre-Mix Working Solution: (Automation) GREEN AP Plus working solution has to be prepared fresh 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 for 10-20 min.

7. Counterstain with Hematoxylin or other counterstain. Wash with DI water followed by immuno wash buffer.
8. Mounting: Slides should be air dried (do not dehydrate in alcohol or xylene). After rinsing off counterstain in DI H2O, leave slides on benchtop for at least 20 minutes to air dry, and then permanently mount.

Note: GREEN AP Plus chromogen-substrate working solution is light sensitive and should be kept away from light as much as possible. Working solution should be prepared fresh in the dark. Recommendation: For best color preservation and long term slide storage, we recommend using Tissue Preservation Solution - HRP/AP assays after counterstaining.