caa a22 4500 475774892 CHVBK 20180406123628.0 cr unu---uuuuu 170329e20000501xx s 000 0 eng 10.1007/s004180000151 doi (NATIONALLICENCE)springer-10.1007/s004180000151 Detection of intracellular nitric oxide using a combination of aldehyde fixatives with 4,5-diaminofluorescein diacetate [Elektronische Daten] [Keiji Sugimoto, Sachiko Fujii, Tohru Takemasa, Kazuo Yamashita] Abstract.: Using 4,5-diaminofluorescein diacetate (DAF-2DA), which was recently developed for the detection of intracellular nitric oxide (NO) in living cells, we examined the sensitivity of intracellular NO in cells treated with some fixatives. Cultured human umbilical vein endothelial cells loaded with DAF-2DA in the presence of 10-6M acetylcholine showed intense fluorescence when fixed in paraformaldehyde or glutaraldehyde, but no fluorescence could be detected after fixation in ethanol or acetone. Fluorescence generation depended on the combination of each aldehyde fixative with DAF-2, which is produced enzymatically from DAF-2DA within the cells. Subtracting the fluorescence intensity of non-activated controls from that of cells activated by acetylcholine indicated the NO produced in the stimulated cells, since the control cells that took up DAF-2DA also generated fluorescence when treated with aldehyde fixatives. Thus, detection of intracellular NO by combining aldehyde fixatives with DAF-2DA is useful for examining the functions of NO in cells both in situ and in vivo. Springer-Verlag, 2000 Nitric oxide Endothelial cells 4,5-Diaminofluorescein diacetate Histochemistry Aldehyde fixatives nationallicence Sugimoto Keiji Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut Fujii Sachiko Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut Takemasa Tohru Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut Yamashita Kazuo Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut https://doi.org/10.1007/s004180000151 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s004180000151 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sugimoto Keiji Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut NATIONALLICENCE 700 1- Fujii Sachiko Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut NATIONALLICENCE 700 1- Takemasa Tohru Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut NATIONALLICENCE 700 1- Yamashita Kazuo Department of Anatomy, Nippon Medical School, 1-1 Sendagi, Bunkyo-ku Tokyo 113-8602, Japan aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer