Oxidized p-phenylenediamine: observations on the staining reaction in epoxy embedded tissues

Abstract

p-Phenylenediamine (PPD) is easily oxidized to brown compounds which stain acidic substrates. On account of the spontaneous oxidation process, the colour of PPD increases and becomes ochre-brown in a few days, showing an absorption peak at lambda = 510 nm with shoulder at about 440 to 460 nm. Studies on the application of oxidized PPD as a stain for semi-thin sections revealed that some tissue components could be clearly visualized. After glutaraldehyde fixation, semi-thin and thin sections of animal tissues were treated with 0.5% aqueous PPD solutions which were aged for variable times at room temperature. Microvilli, goblet cell mucin, mast cell granules, cartilage matrix, collagen, elastin, keratohyalin granules, acrosomes, cytoplasmic granules of Drosophila hydei salivary glands and chromatin showed positive staining reactions after treatment of semi-thin sections with oxidized PPD (7-10 days aged) for 20-30 minutes. Microspectrophotometric studies revealed an absorption peak at lambda = 520-530 nm and a shoulder at lambda = 440-460 nm in goblet cell mucin stained by oxidized PPD. In the presence of anionic macromolecules, the main peak of oxidized PPD solutions showed a strong hyperchromism. Thin sections stained by oxidized PPD did not appear contrasted, but the treatment with 0.125% gold chloride (AuCl3) induced massive gold deposits in structures stained by oxidized PPD. Hyperchromic shifts were also produced in oxidized PPD solutions after the addition of small amounts of AuCl3. This procedure can be used as a simple and rapid staining method for epoxy sections, giving selective contrast for some tissue components.