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1227386 
Journal Article 
Whole-cell immunocytochemical detection of nitrogenase in cyanobacteria: improved protocol for highly fluorescent cells 
Taniuchi, Y; Murakami, A; Ohki, K 
2008 
Yes 
Aquatic Microbial Ecology
ISSN: 0948-3055
EISSN: 1616-1564 
Inter-Research, Nordbuente 23 Oldendorf/Luhe 21385 Germany, [mailto:ir@int-res.com], [URL:http://www.int-res.com/] 
51 
3 (2008) 
237-247 
WOS:000257602400003 
An improved immunocytochemical method was developed to detect nitrogenase in single cells of cyanobacteria using a unicellular diazotrophic strain (Gloeothece sp. 68DGA) and a non-diazotrophic strain (Synechocystis sp. PCC6714) as model organisms. Polyclonal antibodies raised against the Fe- protein and the MoFe-protein (alpha-subunit) of nitrogenase were used as probes. The antigenicity of nitrogenase was maintained for at least 6 mo when the cells were preserved in chilled methanol (-30 degree C) after paraformaldehyde fixation (3%). The cells were permeabilized for antibody penetration and non- specific binding was prevented by incubation in phosphate-buffered saline containing dimethyl sulfoxide (10%) and normal rabbit serum (15%). Antibody binding was visualized by a horseradish peroxidase-conjugated secondary antibody and the chromogenic substrate 3-3'-diaminobenzidine tetrachloride because of the difficulty in discriminating between fluorescence from additive fluorochrome and natural autofluorescence from cyanobacteria. Almost all cells (>97%) were immunostained when they were grown diazotrophically and expressed nitrogenase (acetylene reduction) activity. Non-specific staining of both the diazotrophic strains grown with combined nitrogen and the non- diazotrophic strain was negligible. Our protocol was able to detect nitrogenase in unicellular and filamentous non-heterocystous strains without modification. 
Synechocystis; Antibodies; Nitrogen; Antigenicity; Dimethyl sulfoxide; Nitrogenase; Plant physiology; Phytoplankton; Acetylene reduction; Cyanobacteria; fluorochromes; Methanol; Nutrients (mineral); Cyanophyta; Fluorescence; Staining; Gloeothece; Serum; Probes 
IRIS
• Methanol (Non-Cancer)
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