E. coli BL21(DE3) cells co-expressing Ct-MOMP and Bam were fixed with 0.8% formaldehyde at 4 °C overnight. The cells were gently washed 3x with ice-cold PBST (0.05% Tween 20) and harvested by centrifugation (5000× g, 5 min, 4 °C). To permeabilize membranes and peptidoglycan, the cells were resuspended in PBS, pH 7.4, containing 0.1% Triton X-100, 100 µg/mL lysozyme, 5 mM EDTA and incubated at room temperature for 45 min [66 (link)]
After blocking with PBS containing 3% BSA for 1 h, cells were incubated with primary antisera (anti-MOMP, 1:1000 or anti-BamB, 1:200) for 1 h, subsequently with secondary antiserum (Alexa488 goat anti-rabbit, 1:200) 1 h at RT. Then, cells were immobilized by a thin layered 1% agarose on glass slides. The phase contrast and fluorescence images of cells were obtained by using LSM700 laser scanning confocal system from Zeiss (Florida, USA) and further processed using ImageJ software (http://rsb.info.nih.gov/ij/ ).
After blocking with PBS containing 3% BSA for 1 h, cells were incubated with primary antisera (anti-MOMP, 1:1000 or anti-BamB, 1:200) for 1 h, subsequently with secondary antiserum (Alexa488 goat anti-rabbit, 1:200) 1 h at RT. Then, cells were immobilized by a thin layered 1% agarose on glass slides. The phase contrast and fluorescence images of cells were obtained by using LSM700 laser scanning confocal system from Zeiss (Florida, USA) and further processed using ImageJ software (
Free full text: Click here
