Protocol II: Optotracing of biofilm in tissue sections using EbbaBiolight 680

This protocol describes how to stain for the biofilm markers curli and cellulose in tissue sections. EbbaBiolight can be used to stain tissue sections prepared by the most common techniques like paraffin embedding and freezing. We recommend fixation in ice-cold ethanol, but fixation in 4% paraformaldehyde works as well. We have tested this procedure with Salmonella Enteritidis and Salmonella Typhimurium strains. For these strains we have not observed staining of intracellular or membrane components. As EbbaBiolight is only fluorescent when bound, you can consider to omit washing steps when working with sensitve tissues.

Solutions and Reagents:

EbbaBiolight is provided as 1000-fold concentrated solution. The following common reagents are required (not supplied):

  • Ethanol, 95% (-20°C) or 4% PFA
  • Phosphate buffered saline (PBS), pH 7.4
  • Deionized water
  • Glass coverslips
  • Mounting medium

Assay Procedure:

  • Fix infected cells or tissue sections with method of choice. We recommend fixation with ice-cold ethanol (5 min) at room temperature.
  • Rehydrate tissue sections in a mix of ethanol and deionized water (1:1) for 5 min. The rehydration step may need to be repeated with lower ethanol ratio depending on the tissue.
  • Equilibrate sections in PBS for 5 min.
  • Dilute EbbaBiolight in PBS 1:1000.
  • Apply diluted EbbaBiolight generously. Use enough liquid (ca 0.5 ml) to prevent the sections from drying out during incubation. Incubate for 30 min.
  • Wash 2 x 5 min in PBS (Optional).
  • Mount tissue sections and seal the coverslip onto the slide to prevent drying.

Fluorescence Microscopy:

  • EbbaBiolight 680 is excited at 561 nm (standard laser line) and emission is detected using a standard PI (Propodium Iodide), mCherry or Cy3.5 filter set. Optional: An excitation range of 530-565 nm and a detection range of 600-800 nm may be applied depending on available laser lines and filter sets.

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