Mercuric Bromphenol Blue Staining of Precipitin Lines in Agar

Abstract

Lines formed by antibody-organ antigen reactions are stained particularly well by a modification utilizing the mercuric bromphenol blue (MBB) mixture of Mazia et al. (Biol. Bull., 104: 57-67, 1953). The agar covered slides are placed overnight in 0.85% NaCI at 4 C, followed by washing for 2 hr in 0.85% NaCI at 25 C. They are then rinsed for 10 min in distilled water, and dried overnight at 37 C. The precipitin lines are fixed by immersing the slides for 25 min in 95% alcohol, followed by 5 min hydration in distilled water. They are stained for 25 min in MBB mixture (HgCI₂, 10 gm; bromphenol blue, 0.1 gm; 95% ethanol, 100 ml). Excess stain is removed by immersing in acidified alcohol (95% ethanol, 98 ml; glacial acetic acid, 2 ml). Finally, the slides are passed through alcohol and xylene, and resin-mounted under coverslips.