Poor contrast in a hematoxylin and eosin stain occurs when nuclear staining is partially masked by the eosin.
Causes of poor contrast include:
(1) nuclear stain is too pale
(2) cytoplasmic stain is too dark
(3) cytoplasmic stain is too pale
Solutions to improving contrast:
(1) adjust the time spent in the hematoxylin solution to increase or decrease stain intensity
(2) maintain a proper pH in the hematoxylin stain (pH 2.3 to 2.7)
(3) make sure that the rinsing steps are effective in removing excess hematoxylin stain
(4) monitor the rinsing water pH and make sure that it is relatively constant
(5) adjust the time spent in eosin stain to increase or decrease stain intensity
(6) maintain a proper pH in the eosin stain (ph 4.0 to 4.5)
(7) adjust the percentage alcohol in the wash steps and/or the rinsing times
