Steps in an H&E Stain

H&E staining is a process. Learning the steps in H&E staining is essential for every new tech, but there is more to the H&E staining process than just completing the steps in order. What do you do if you miss a step? What do you do if you perform a step incorrectly?

The good thing about the H&E staining process is that you can work your way backwards through the steps to correct mistakes. For example, if you have over stained with your hematoxylin, you can use your differentiator to pull out the hematoxylin, rinse in running water, and redo the hematoxylin step. The important thing to remember however, is that you cannot skip steps. You can’t go backwards from water to xylene without your EtOH step, as water and xylene are not miscible.

There are also several steps that can serve as holding steps; steps where it is safe to leave the stain to go answer a phone or perform another lab task. Knowing these hold steps can help you avoid having to redo steps and work backwards.

The bolded steps in the graphic shown here are okay to hold. You will notice xylene and running water are okay throughout. Step 8 and 16, the 70% EtOH steps are also okay to use as a holding step, as this is the right balance of water that our cells have, so our tissue won’t swell or shrink. You cannot use the step 18, 70% EtOH step as a holding step however, because this step is used as the differentiation step for the Eosin and start of dehydration. It would remove way too much eosin if it is left in.

But what about the bluing step? Yes, it is true that you can’t “over blue” the slide (Check out a previous blog post on bluing agents!), but it is not recommended that you use the bluing step as a holding step, primarily because too long in a harsh bluing reagent such as ammonia solution can make sections fall off. If you miss the bluing step you can move backwards through the steps from xylene to alcohol to water and then do the bluing and continue the steps forward. Alternatively, depending on the pH of your water, you may be able to get away with rinsing in tap water for a few minutes and achieve a bluing effect, but the bluing step will enhance the blue color of the hematoxylin.

If you're interested in learning more, you can find more info in NSH's HT Prep Course, a series of webinars covering basic histology exam topics.

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