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In the attempt to develop a pre-embedding immunoelectron microscopic method with improved ultrastructral morphology, we examined an antigen retrieval (AR) method using citraconic anhydride, and compared the effects of glutaraldehyde fixation with routine paraformaldehyde fixation in the pre-embedding immunoelectron microscopic method with reference to the localization of surfactant-associated pro-protein C (proSP-C) in the lung. The glutaraldehyde-fixed tissues were immunostained after AR in 0.05% citraconic anhydride solution, pH 7.4, at 98 degrees C for 60 min. In routine pre-embedding immunoelectron microscopic method using paraformaldehyde fixation, proSP-C positive products were distributed sporadically in the type II alveolar epithelial cells. In glutaraldehyde-fixed tissues without the AR method, proSP-C products were not detected, however after AR in citraconic anhydride proSP-C positive products were distributed specifically, in rough endoplasmic reticulum membranes, Golgi complex membranes, multivesicular bodies and osmiophilic lamellar bodies. The positive proSP-C products also showed lattice-like structures in the alveoli. Thus, the present AR method provides successful pre-embedding immunoelectron microscopy of glutaraldehyde-fixed tissues.