The Combination of Fixation Using PLP Fixative and Embedding in Paraffin by the AMeX Method is Useful for Immunohistochemical and Enzyme Histochemical Studies of the Lung

To establish a method of lung processing suitable for morphological, immunohistochemical and enzyme histochemical analyses, we examined the combination of inflated-fixation with periodate-lysine-paraformaldehyde (PLP) fixative and embedding in paraffin by the AMeX method. The lungs were removed from rats with or without bleomycin (BLM) treatment and the airways were instilled with PLP fixative. After inflated-fixation, the specimens were processed and embedded in paraffin by the AMeX method. Paraffin sections were stained immunohistochemically and enzyme histochemically. Hematoxylin and eosin (HE)-stained sections were also subjected to evaluation of tissue architecture. In the HE-stained sections, the bronchioles, alveolar ducts, and alveolar sacs were uniformly inflated, and the architecture was well preserved. In addition, detailed histopathological findings of lung lesions induced by BLM could be observed. In immunohistochemical staining, all of the antibodies used in this study (anti-CD3, -CD45RA, -ED-1, and -alpha-smooth muscle actin monoclonal antibodies) stained the cells positively according to their specificities. It is noteworthy that CD3 and CD45RA, which are not detected in routinely formalin-fixed paraffin sections, were clearly detectable. In enzyme histochemical staining, alkaline phosphatase activity was very well preserved. These findings suggest that the present method is useful for immunohistochemical and enzyme histochemical analyses of the lung.