BLACK HRP Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. Aliquot 1mL of BLACK HRP Substrate Buffer in a mixing bottle
2. Add one drop (~20ul) of concentrated BLACK HRP Chromogen solution.
3. Replace tip, mix, and allow solution to reach room temperature before using.
4. Working solution is stable for up to 1 day, for optimal results prepare fresh reagent.

Note: The BLACK HRP 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 1 day, for optimal results prepare fresh reagent.

Staining Procedure
1. Once sections have been incubated with peroxidase, wash sections with wash buffer then follow protocol of choice:a. Pre-Mix Working Solution: (Automation) BLACK HRP has a working solution stability of at least 1 day 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 5-10 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 in on-board mixing station before application to slide. Incubate for 5-10 minutes.
c. Manual Use Mix substrate-chromogen and buffer in a 1:50 ratio and apply directly to slide. Incubate for 5-10 minutes.2. Counterstain with Hematoxylin or other counterstain.
3. Wash with DI water followed by immuno wash buffer.
4. Slides can be dehydrated through alcohol and xylene or xylene substitute or can be air dried, then permanently mounted.
Recommendation: For best color preservation and long term slide storage, we recommend to use Tissue Preservation Solution - HRP/AP assays after counterstaining.