Combination of fixation using PLP fixative and embedding in paraffin by the AMeX method is useful for histochemical studies in assessment of immunotoxicity

Abstract

To establish a method for processing lymphoid organs suited to morphological, immunohistochemical and enzyme histochemical analyses for assessment of immunotoxicity, we examined a combination of fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method (PLP-AMeX method). Spleen and thymus removed from monkeys and rats were fixed in PLP fixative for 6 hours at 4°C. After fixation, specimens were processed and embedded in paraffin by the AMeX method. In hematoxylin and eosin-stained sections, tissue architecture was well preserved. In immunohistochemical staining, markers of T lymphocytes (CD3, CD4, CD8), B lymphocytes (monkey: CD20cy, rat: CD45RA) and macrophage (monkey; CD68, rat: ED-1) were well identified according to their specificities, although the staining intensity of CD8 in the monkey and CD4 in the rat were somewhat weaker in PLP-AMeX-prepared sections than in those frozen. In enzyme histochemical staining, alkaline phosphatase activity was well preserved in neutrophils. In toluidine blue- and Giemsa-stained sections, eosinophil granules and the metachromasia of granules in basophil/mast cells were clearly detectable. These findings suggest that the PLP-AMeX method is a powerful tool for assessment of immunotoxicity.