Immunofluorescent Analysis of Trout Gut and Skin Bacteria

Bacterial pellets isolated from healthy rainbow trout (N = 3) gut and skin mucus obtained as explained elsewhere5 (link)10 . Bacterial suspensions were placed onto sterile microscope slides and were immediately blocked with T20 protein blocking solution (ThermoFisher) for 15 min. Slides were incubated with primary anti-trout pIgR antibody or the prebleed antibody as a negative control, followed by Cy2-conjugated donkey anti-rabbit secondary antibody (Jackson Immunoresearch). Slides were then stained with a solution containing DAPI (1 μg/mL) and Hoescht (2 μg/mL) DNA stain for 30 min, rinsed in tap water, and mounted with fluorescent mounting media. Slides were observed under a Nikon Ti fluorescent microscope, and images acquired with the NIS Advanced Research Software v.4. A total of ten images (x60) per sample were captured, and the numbers of Cy2⁺ and Cy2⁻ bacteria were counted in each image.

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Kelly C., Takizawa F., Sunyer J.O, & Salinas I. (2017). Rainbow trout (Oncorhynchus mykiss) secretory component binds to commensal bacteria and pathogens. Scientific Reports, 7, 41753.

Publication 2017

Cy2-conjugated donkey anti-rabbit secondary antibody

Anti antibody Antibody Bacteria Dapi Donkey Microscope Mucus Pellets Protein Rabbit Rainbow trout Skin Stain Sterile Trout

Corresponding Organization :

Other organizations : University of New Mexico, University of Pennsylvania

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Variable analysis

independent variables

Bacterial pellets isolated from healthy rainbow trout gut
Bacterial pellets isolated from healthy rainbow trout skin mucus

dependent variables

Number of Cy2+ bacteria
Number of Cy2- bacteria

control variables

Bacterial suspensions were placed onto sterile microscope slides
Slides were immediately blocked with T20 protein blocking solution for 15 min
Slides were incubated with primary anti-trout pIgR antibody or the prebleed antibody as a negative control
Slides were stained with a solution containing DAPI (1 μg/mL) and Hoescht (2 μg/mL) DNA stain for 30 min
Slides were rinsed in tap water, and mounted with fluorescent mounting media
Slides were observed under a Nikon Ti fluorescent microscope, and images acquired with the NIS Advanced Research Software v.4

positive control

Primary anti-trout pIgR antibody

negative control

Prebleed antibody

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