Staining of Small Lymphoid Nucleoli in Paraffin Sections by Aniline-Azure B

To see small lymphoid nucleoli clearly in 1-2 μ paraffin sections, the staining of contiguous chromatin masses in the nucleus was suppressed by a hydrolysis-aniline blocking sequence, which produces aldehyde from DNA, and attaches aniline to that aldehyde to make a diphenamine base, thus reducing the acidity of the chromatin and its affinity for basic dyes. Nucleolar RNA remains fully stainable by azure B, because the hydrolysis used does not produce aldehyde groups in it, to allow aniline attachment. Technique: Hydrolyse the 10% formol-saline fixed, deparaffinised 1-2 μ section for 4.5-5.0 min in 10% (v/v) HCl in tetra-hydrofuran at 39-40 C, rinse in water, and treat at room temperature in 10% (v/v) aniline in acetic acid for 10 min. Stain 2-4 hr with freshly prepared 0.1% azure B in a 1:10 dilution of tris buffer at pH 7.0. Rinse, blot off excess water, pass through acetone and xylene to a polystyrene mounting. DNA stains pale green to colourless; nucleolar and cytoplasmic RNA, blue.