Troubleshooting Problems with Nuclear Staining with the Trichrome Stain

Problems:

(1) pale staining

(2) poor nuclear detail

(3) overstaining (nuclei too dark with indistinct chromatin)

Pale staining can occur if:

(1) A hematoxylin stain other than iron hematoxylin was used.

(2) The iron hematoxylin stain was not made fresh, with oxidation of the stain.

(3) Time in the nuclear stain was inadequate.

(4) The red and blue counterstains were poorly differentiated.

Poor nuclear detail or overstaining can occur if:

(1) The section was overstained in the iron hematoxylin solution.

(2) The slide was not adequately washed (rinse in 70% ethanol then water) after staining in the iron hematoxylin.