Effect of pH on hematoxylin and eosin staining of rats intestinal tissue

Background: Optimising hematoxylin and eosin (H&E) protocol does not only ensure reproducible and excellent staining quality, it also helps to optimise the use of lab materials and resources. This study investigates the effect of hematoxylin pH on H&E staining quality. Method: In an experimental study, 3 μm tissue sections of formalin-fixed paraffin embedded intestinal tissues of three healthy female Sprague-Dawley rats were subjected to the standard H&E protocol. Modified Gill’s hematoxylins of four pH (2.5, 2.75, 3.0 and 3.5) and eosin (pH 5.0) as counterstain were used. Results: At hematoxypin pH 2.5, the tissues appeared acidophilic with indistinctive epithelial lining. At pH 2.75, the hematoxylin dye was most prominent with good balance of staining, and crisp epithelial lining was observed. At pH 3.0 or higher, although the lining was crisp, blue-staining mucin were observed. Conclusion: pH 2.74 is the most optimal pH to achieve balance of coloration and definitive epithelial lining in H&E staining.