An improved method for Gill's hematoxylin and eosin staining based on the optimal acetic acid ratio

The staining performance of hemalum solutions is critically dependent on pH, yet the acetic acid content varies among different hemalum formulations. This study aimed to clarify the relationship between the acetic acid ratio (pH) of Gill's hemalum and optimal nuclear staining, and to improve the hematoxylin and eosin (H&E) staining method. Gill's hemalum with varying acetic acid concentrations was used for H&E staining of surgical and biopsy specimens. Optimal nuclear staining was achieved at 3.48% acetic acid (pH 2.47), with pH deviations compromising staining quality. Tissue-specific differences in nuclear staining were observed: liver, prostate, and lung stained better than stomach, thyroid, and breast, and surgical specimens outperformed biopsies. Notably, the staining effect of overacidified Gill's hemalum could not be restored by adding sodium hydroxide (NaOH). At the optimal pH, Gill's hemalum surpassed Harris' hemalum in nuclear staining quality. Furthermore, the addition of a pH buffer not only improved staining consistency but also significantly extended the usable lifespan of Gill's hemalum. These findings indicate that the acetic acid concentration (pH) of Gill's hemalum is a key quality control parameter for H&E staining. Gill's hemalum prepared with 3.48% acetic acid and used with a pH buffer, offers significant practical value and is recommended for routine use in a pathology laboratory.