If there is understaining with eosin then the cytoplasmic staining of an H&E section will be too light.
Conditions associated with eosin understaining:
(1) problem with eosin stain (poor quality, exhausted, wrong concentration)
(2) insufficient time in the eosin solution
(3) pH of the eosin solution too high (above 4.5)
(4) carry-over of bluing solution
(5) prolonged or improper rinsing of the slide in alcohol after eosin staining
Steps to correct:
(1) Check the time spent in each solution.
(2) Replace the working eosin solution.
(3) Check the quality of the eosin stock solution.
(4) Check the pH and adjusted with concentrated acetic acid if too high (target 4.0 to 4.5).
(5) Make sure that the bluing reagent has been completely removed prior to eosin step.
(6) Check the quality of the alcohol wash solutions after staining in eosin.
