Antifade

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.

For the confocal imaging studies, Vero cells in 35 mm dishes were washed twice with 1 × PBS. Cells were then treated with Cu(NO₃)₂ in DMEM and incubated at 37 °C in a CO₂ incubator for 2 h. After 2 h, the cells were again washed twice with 1 × PBS and incubated with 25 μM HL in DMEM at 37 °C for 30 min in a CO₂ incubator. The cells were then washed twice with 1 × phosphate buffered saline (PBS) (pH 7.4) and fixed with 4% paraformaldehyde (w/v) for 15 min at room temperature. Subsequently, the cells on the coverslips were washed twice with 1 × PBS and the coverslips containing fixed cells were mounted onto a glass slide with a mounting medium containing antifade (Invitrogen) to reduce photobleaching. Fluorescence microscopic images were obtained using a Leica TCS SP5 confocal microscope using a 40× objective at λ_ex = 405 and λ_em = 466 nm. All the images were analyzed using Leica Application Suite Advanced Fluorescence software (LASAF).^{44 (link)}

Cells were grown on coverslips, washed twice with 1X PBS and fixed with 4 % paraformaldehyde for 10 minutes at 37ºC. The cells were quenched using 0.1 M Glycine in 1XPBS for 10 minutes, permeabilized using 0.1 % Triton X-100 in PBS for 5 min and blocked with 1 % fetal bovine serum (FBS), 1 % bovine serum albumin (BSA) in PBS for 45 minutes. Primary staining was conducted in the blocking solution for one hour at room temperature, washed with 1XPBS-Tween 3 times for 5 minutes, and incubated with secondary in blocking solution for 1 hour. The coverslips were rinsed 3 times for 10 minutes each with 1XPBS-Tween and once with Millipore water then mounted using Anti-Fade (Invitrogen). Images were acquired on a Zeiss Axio Observer 7 with an ApoTome.2 for inverted fluorescence microscopy.