Fixation on Histology

Troubleshooting H&E Stains


Troubleshooting H&E StainsThe H&E stain is often described as the histologist’s bread and butter stain, as it is used so often and is the foundation for histology staining education. That may be the case, but that doesn’t mean that troubleshooting it is easy. There are numerous variables that can affect the quality of your H&E stain. In this post we will cover some common problems you may run into.


The slide is too pink.

If the slide is too pink it is generally a result of your eosin and can be solved by altering the time that you leave the slide in various steps within the H&E staining process. If your eosin is too strong you may need to decrease the time that you’re leaving the slide in it or increase the time that it is left in the 95% alcohol afterwards. Varying the percentage of alcohol that you use before and after eosin can also change the pinkness. The more water there is in the alcohol following the eosin, the more eosin there will be removed from the tissue.


If the slide is a reddish color it can be due to inadequate blueing. It is not possible to over-blue a slide, but you can under blue it. The blueing reagent changes the hematoxylin to a blue/purple color. This reaction is pH dependent, so make sure that your blueing reagent has a pH of approximately 8.


Staining is too light.

A common factor to check here is the pH of your water. We previously did a post on how the pH of water will impact your H&E staining. If you’re using tap water, changes from the city can cause seemingly random changes to the quality of your staining. You can also try adding a few drops of glacial acetic acid to your eosin. You will also want to check your machinery to make sure that reagents are being changed on a schedule that is appropriate for the volume you’re processing. You can also check your rinse times to make sure that the tissue is not left in rinse too long, giving it a washed out appearance. Another potential cause is incomplete deparaffinization. If paraffin is not totally removed it can make the staining appear light or spotty.


As with all troubleshooting, if you’re not finding an immediate solution to your problem, methodically check other factors; everything from your tissue adhesive, to your oven, to hematoxylin beyond its shelf life could be impacting your staining.


Slides are hazy or milky.

After eosin staining, increasing concentrations of alcohol are used to remove water from the tissue section. Inadequate dehydration (removal of non-anhydrous (70%, 95% alcohol) from the tissue section will cause slides to be hazy or milky. The haziness/milkiness of the slide is usually due to the water mixing with the xylene. When using denatured alcohol, be mindful of the presence of denaturants that are not water soluble in the alcohol as this can also cause a milky appearance.