StayBlue/AP is a substrate chromogen system designed to be used for either IHC or ISH when using alkaline phosphate detection. StayBlue/AP produces a vibrant blue color. It is insoluble in alcohol and xylene solutions (both aliphatic and hydrocarbon based) therefore sections can be dehydrated in alcohol, cleared in xylene substitute and permenently mounted.

Working Solution

Aliquot 3ml of StayBlue/AP substrate buffer in a mixing bottle. Add 20µl (one drop) of concentrated StayBlue/AP chromogen. Replace tip, mix and allow the solution to reach room temperature before using.
Note: The working chromogen-substrate solution should be prepared fresh and used withing 25 mins of preparation. Any solution not used during this period should be discarded.

Protocol

• After SA-AP or AP-polymer incubation, wash tissue sections with wash buffer.


• Wipe slides removing excess buffer. Add enough drops of StayBlue/AP working solution to cover tissue sections.


• Incubate for 5-25 minutes at room temperature. For optimal results, observe reaction under the microscope for signal development. Once the desired signal to noise ratio is achieved, stop the reaction by rinsing the slides with DI H₂O.


• Note: Increasing incubation temperature to 37°C will increase sensitivity and decrease needed incubation time.


• Counterstain. Nuclear Fast Red provides good contrast.


• Wash with DI H₂O.


• Dehydrate sections in alcohol, clear xylene substitute and mount in permanent mounting medium.


• Note: Use increasing concentrations of ethanol (up to 100%) to dehydrate. Use xylene substitute instead of xylene.


• Alternatively, slides can be air dried. After rinsing off counterstain in DI H₂O, leave the slides on the benchtop for at least 20 minutes to air dry, and then permanently mount.

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