Leukemia inhibitory factor (LIF) secretion in primary astrocytes is constitutive and independent of NECA stimulation. (A) illustrates LIF immunocytochemistry in cultured cortical astrocytes, where LIF is found in vesicle-like structures throughout the cytoplasm. Scale bar corresponds to 10 μm. (B) shows complete co-localization of LIF with Giantin, a marker of Golgi apparatus, when cultured astrocytes were treated with Brefeldin A (BFA, 5 μg/mL for 1 hour). Scale bar corresponds to 10 μm. (C) shows a representation of sandwich ELISA experiment performed to detect LIF content in supernatants of cultured astrocytes that were treated without or with NECA (1 μM) for 4 hours. Where indicated, cells were pre-treated with BFA (5 μg/mL) for 1 hour prior to NECA stimulation. Each bar corresponds to the mean concentration of LIF in triplicate samples; error bars indicate SEM. Observations were confirmed by repeating the experiments two additional times. P < 0.05. NECA, 5′-N-ethylcarboxamide.