YELLOW HRP Staining Kit (Colorimetric)

Prepare the Following Solutions Before Use
1. Aliquot 1mL of YELLOW HRP Substrate Buffer in a mixing bottle.
2. Add one drop (~20ul) of concentrated YELLOW HRP Chromogen solution.
3. Replace tip, mix, and allow solution to reach room temperature before using.
4. The YELLOW HRP chromogen-substrate working solution is light sensitive and should be 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) YELLOW 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 10-20 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 10-20 minutes.
c. Manual Use Mix substrate-chromogen and buffer in a 1:50 ratio and apply directly to slide. Incubate for 10-20 minutes.

2. Counterstain with Hematoxylin or Nuclear Fast Red for good contrast.
3. Wash with DI H2O followed by immuno wash buffer. Wash with DI water followed by immuno wash buffer.
4. Slides should be air dried (do not dehydrate in alcohol).
5. After rinsing off counterstain in DI H2O, leave slides on benchtop for at least 20 minutes to air dry.
6. Then permanently mount.
Recommendation: For best color preservation and long term slide storage, we recommend using Tissue Preservation Solution - HRP/AP assays after counterstaining.