Computational protocol: Dynamic nature of SecA and its associated proteins in Escherichia coli

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Protocol publication

[…] Indirect immunofluorescence microscopy of E. coli cells was performed as described previously (Hiraga et al., ; Hirano et al., ). Rabbit anti-SecA/SecY antisera were gifts from Dr. Koreaki Ito (Kyoto Sangyo University). Rabbit anti-AcpP antiserum was a gift from Dr. Charles Rock (St. Jude Children's Research Hospital). Mouse anti-GFP monoclonal antibody is commercially available (Roche Diagnostics, K.K., Tokyo, Japan). We used goat anti-rabbit/mouse IgG antiserum conjugated to Cy3 (GE Healthcare, U.K. Ltd., Little Chalfont, UK) as the secondary antibody. For optical sectioning, an OLYMPUS BX61 microscope with an OLYMPUS UPlanApo ×100/1.35 oil objective lens (OLYMPUS, Corp., Tokyo, Japan) connected to CoolSNAPHQ (NIPPON ROPER, K.K., Chiba, Japan) was equipped with MetaMorph (Universal Imaging, Corp., Downingtown, PA) to obtain series of Z-sections with a fixed spacing of 0.1 μm. Each stack of 26 optically sectioned fluorescent images was deconvoluted through 50 iterations using the three-dimensional deconvolution function in the AutoQuant X version 1.4.1 program (Media Cybernetics, Inc., Silver Spring, MD). Three-dimensional images were then obtained by Volume Viewer function of 3D plug-in in ImageJ software. […]

Pipeline specifications

Software tools MetaMorph, AutoQuant, ImageJ
Application Microscopic phenotype analysis
Organisms Escherichia coli