Iplab

The locations of bacteria and ZsGreen in tumors was determined with immunofluorescence. Paraffin-embedded tumor sections were rehydrated and treated with sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 20 min at 65°C for antigen retrieval. Sections were blocked with protein block (Dako, Santa Clara, CA); stained with two primary antibodies, 1:100 rabbit anti-RCFP polyclonal antibody (Clontech) and 1:10 FITC-conjugated, anti-Salmonella antibody (Abcam, Cambridge, MA); and washed with TBS-T. A secondary antibody, Alexa Fluor 546-donkey-antirabbit IgG (Life Technologies, Carlsbad, CA), was applied at 1:100. 4′,6-Diamidino-2-phenylindole (DAPI) was applied to stain cell nuclei and identify necrosis. Immunofluorescent images were acquired with an inverted epifluorescent microscope (Olympus, Tokyo, Japan) with a 10x Plan-APO fluorescence objective and an automated script written in IPLab (BD Biosciences, Rockville, MD). The script assembled a tiled montage of individual images acquired using three fluorescent filters (Chroma, Bellows Falls, VT): D350/50x (DAPI), D546/10x (ZsGreen) and D455/70x (Salmonella).

Representative pGluN1 signals (Fig. 2A1) were manually selected (red circles in Fig. 2A2), and the area and intensity of the fluorescent signal were measured in each red circle using image analysis software (IPLab, BD Bioscience, San Jose, CA). The range (max/min) of areas and the range (max/min) of fluorescent intensities measured from a total of 236 red circles were used to establish selection criteria for pGluN1 immunoreactive puncta in 30 slices. The criteria, once established, were applied to immunofluorescent signals that were acquired from a total of 2024 images taken from 157 slices in 11 pups using auto-segmentation logic (Triangle), and only those that satisfied the criteria were accepted as pGluN1 immunoreactive puncta (in 512 images in 114 slices in 11 pups). pGluN1 immunoreactivity was quantified as a ratio between the summed total area of pGluN1 immunoreactive puncta over a total image area, and presented graphically as mean ± SEM (standard error of the mean) according to statistical methods set by the EJN Authors' Guideline (EJN vol. 28, pp. 2363-2364). Results were tested for statistical significance with a student t-test or ANOVA (analysis of variance). Differences were considered significant if P < 0.05.