In a previous post, we talked about the importance of knowing the pH of your water in an H&E stain. (Click here if you missed it!). In summary, your pH matters because the reaction that takes place during an H&E stain is pH dependent and needs to occur in an alkaline solution.
In this case, water is acting as the bluing reagent in the H&E stain. A bluing reagent can be water, but it can also be an ammonia solution, Scott’s solution, or lithium carbonate solutions. The purpose of a bluing reagent is to… duh… make it blue!
More specifically, the bluing reagent changes the reddish-purple hematoxylin to a blue or purple-blue color. If you are troubleshooting an H&E stain and you notice the stained section is a reddish color, that means the bluing reagent didn’t do its job, potentially because the pH was too low, and the reaction did not properly occur.
Alternatively, it isn’t possible to over-blue a section. The bluing reagent will blue the amount of hematoxylin in the tissue so if your section is too blue, there was too much hematoxylin in the tissue. You should be cautious however about leaving a section too long in a harsh bluing reagent such as ammonia solution since it can make sections fall off.
Because of the impossibility of “over-bluing” it is relatively easy to tell when the bluing reaction has occurred. Generally, for water or other aqueous solutions, this process will take about a minute, while solutions such as the ammonia solution will take less time.