Localization of Bacteria in Epithelial Tissues

Pieces of tongue and stomach epithelia were collected and sectioned in a sterilised setup. Sections were fixed in 1:1 acetone-to-methanol and incubated with PNA FISH probes at 55 °C. A universal bacteria probe (BacUni; AdvanDx) and probe for C. dubuliniensis (AdvanDx) were used^{25 (link)}. DAPI was used for nuclear counterstaining. Slides were mounted using ProLong Gold anti-fade reagent (Life Technologies). Images and Z-stacks were acquired with a Nikon A1 Upright Confocal microscope. Evaluation of bacterial location was performed on three-dimensional z-stacks using ICY software.

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Sequeira I., Neves J.F., Carrero D., Peng Q., Palasz N., Liakath-Ali K., Lord G.M., Morgan P.R., Lombardi G, & Watt F.M. (2018). Immunomodulatory role of Keratin 76 in oral and gastric cancer. Nature Communications, 9, 3437.

Publication 2018

ProLong Gold anti-fade reagent A1 Upright Confocal microscope

Acetone Bacterial Confocal microscope Dapi Epithelia Fish Gold Methanol Stomach Tongue

Corresponding Organization : Guy's Hospital

Other organizations : Howard Hughes Medical Institute, Stanford University

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Variable analysis

independent variables

Tissue type (tongue and stomach epithelia)

dependent variables

Bacterial location (evaluated on three-dimensional z-stacks)

control variables

Sterilized setup for tissue collection and sectioning
Fixation of sections in 1:1 acetone-to-methanol
Incubation temperature of 55 °C for FISH probes
Use of DAPI for nuclear counterstaining
Mounting of slides using ProLong Gold anti-fade reagent

controls

Positive control: Universal bacteria probe (BacUni)
Specific control: Probe for C. dubuliniensis

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