Intended Use:

Substrate/chromogen in conjunction with HRP-based immunostaining or in situ hybridization systems.  With at least 1 day working stability, this substrate/chromogen system can be used in any automation system as well as for manual use.

Working Solution:

Aliquot 1 mL of StayYellow/HRP Substrate Buffer into the mixing bottle. Add one drop (~20 μL) of concentrated StayYellow/HRP Chromogen solution. Replace tip, mix, and allow solution to reach room temperature before using.

Note: The working chromogen-substrate solution is light sensitive and should be kept away from light as much as possible.  The working solution is stable for up to 1 day, however for optimal results prepare fresh reagent.

Staining Procedure: 

Following peroxidase incubation, wash tissue sections with wash buffer then follow the protocol of choice

• Pre-Mix Working Solution: (Automation):

StayYellow/HRP has a working solution stability of at least 1 day and can be loaded directly onto the instrument as a single solution. Reduce exposure to light to achieve optimal staining. Working solution is applied directly to slide.  Working Solution:10-20 minutes

• On Board Mixing (Automation):

Instruments that have on-board 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.  Working Solution:10-20 minutes

• Manual Use:

Mix substrate-chromogen and buffer in a 1:50 ratio and apply directly to slide.  Working Solution:10-20 minutes

Counterstain:  

Counterstain with Hematoxylin or Nuclear Fast Red for good contrast.  Wash with deionized water followed by immuno wash buffer.

Mounting:

Slides should be air dried (do not dehydrate in alcohol or xylene).  After rinsing off counterstain leave slides on benchtop for at least 20 minutes to air dry, then permanently mount.