Axiovision fluorescence microscope

Intracellular location of Hp0897 and its colocalization with HpDnaB were investigated using previously described protocols (37 (link),38 (link)). Rabbit anti-HpDnaB (1:1000) (11 (link)) and mice anti-Hp0897 (1:500) were used as primary antibodies. In brief, H. pylori cells were smeared on poly-lysine (0.01%) coated glass slides and fixed with 4% paraformaldehyde in 1× phosphate buffered saline (PBS) for 15 min at room temperature followed by washing with 1× PBS for three times and treated with Triton-X100 (0.3% in 1× PBS) for 25 min at 25°C. Subsequently, the slides were washed (1× PBS) and blocked (3% bovine serum albumin (BSA) in 1× PBS) for 1 h. Primary antibodies treatment (1:1000 dilutions in 1× PBS containing 3% BSA) for anti-HpDnaB (in rabbit) and (1:500 dilutions in 1× PBS containing 3% BSA) for anti-Hp0897 (in mice) were done at 25°C for 1 h or at 4°C overnight. After washing (1× PBS), the cells were incubated with secondary antibodies (1:1000 dilutions for Alexa fluor 594 conjugated anti-mice IgG antibodies and 1:1000 dilutions for Alexa fluor 488 conjugated anti-rabbit IgG antibodies) obtained from Santa Cruz, CA, USA. Cells were further washed (1× PBS) and mounted with antifade (Invitrogen). An AxioVision fluorescence microscope (Nikon) was used to capture the images. AxioVision, release 4.6 (Nikon) software was used for analysis of the images.