Methylene Blue Staining of Axons in the Ventral Nerve Cord of Insects

Axons and some nerve cell bodies in the abdominal nerve cords of 5 species of insects were stained within 0.5-2 hr after intraabdominal or intrathoracic injection of a rongalit-reduced 0.4% methylene blue solution at pH 5. Leuco methylene blue solutions produced by Na₂S₂O₄, or by rongalit at a lower pH, were not as effective. Injection of the stain into an intact animal produced much better results than application to a dissected preparation. The stain was fixed with a cold, about 1.5% ammonium picrate solution followed by cold 8-15% ammonium molybdate. The nerve cord was removed, placed on a slide, dehydrated with t-butanol, cleared with xylene, and covered.