The SignalStain^® Ultra Blue Alkaline Phosphatase Substrate Kit contains all of the necessary reagents to prepare a working solution of Ultra Blue for staining tissue sections. The Ultra Blue working solution is compatible with alkaline phosphatase (AP) detection systems such as the SignalStain^® Boost IHC Detection Reagents (AP, Rabbit #18653, AP, Mouse #31926, AP, Rat #15764, and AP, Goat #26927), yielding a heat stable, blue reaction product that is also fluorescent.

1. To prepare the Ultra Blue/AP chromogen-substrate working solution, add 1 drop SignalStain^® Ultra Blue Chromogen Concentrate to 1 mL SignalStain^® Ultra Blue Diluent. Mix well before use. Working solutions are stable for up to 1 day when stored at 4ºC. For optimal results, prepare fresh reagent.

2. After incubation with SignalStain^® Boost IHC Detection Reagent or other AP-based detection systems, wash slides and incubate with the working solution at room temperature until staining develops. 8-20 minutes generally provides an acceptable staining intensity.

3. Wash slides for 5 minutes in water.

4. Counterstain, if desired. Note that Hematoxylin #14166 is unlikely to provide sufficient contrast with Ultra Blue.

5. Mount coverslips. After rinsing off the counterstain, air dry the slides for 20 minutes or longer. Permanently mount coverslips with SignalStain^® Aqueous Mounting Medium #27290. Alternatively, slides may be dehydrated through graded alcohols and cleared in a xylene substitute, then mounted with a permanent mounting medium, SignalStain^® Mounting Medium #14177. Ultra Blue is not compatible with xylene.

For best color preservation and long-term slide storage, it is recommended to use SignalStain^® Mounting Prep Solution #50815 after counterstaining.

Appropriate personal protective equipment should be worn when handling Ultra Blue. Dispose of in accordance with local regulations.