Biotin was used as a tracer molecule to determine the integrity of the epithelial barrier20 (link). Briefly, immediately after removal of the entire intestinal tract from mock, C227 or Δstx2 infected rabbits, the colonic tissue was separated while maintaining their correct orientation (ie proximal vs distal ends). EZ-Link Sulfo-NHS-Biotin (Thermo Scientific, IL) was injected slowly (1–2 min) into the lumen via the open (cut) end of the most distal part of the colon. After 3 min, the tissue section just proximal to the site of injection was removed, fixed in 4% paraformaldehyde and processed for immunofluorescence staining as described above. Tissue sections were incubated with streptavidin linked to Alexa 546 (1/500) (S11225; Invitrogen) for 1 hr at room temperature before being counterstained with phalloidin Alexa fluor 633 and DAPI. Colonic tissue from mock, C227 or Δstx2 infected rabbits that were not treated with biotin exhibited no background staining following incubation with streptavidin only.
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Intestinal Epithelial Barrier Integrity Assessment
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Biotin
Colonic
Dapi
Immunofluorescence
Intestinal tract
Paraformaldehyde
Phalloidin
Rabbits
Streptavidin
Sulfo nhs biotin
Tissue
Corresponding Organization :
Other organizations : Brigham and Women's Hospital, Harvard University, Icahn School of Medicine at Mount Sinai
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Variable analysis
independent variables
- Infection status of rabbits (mock, C227, or Δstx2)
- Integrity of the epithelial barrier
- Orientation of the colonic tissue (proximal vs. distal ends)
- Time between biotin injection and tissue removal (3 minutes)
- Positive control: Colonic tissue from mock, C227 or Δstx2 infected rabbits treated with biotin and stained with streptavidin-Alexa 546.
- Negative control: Colonic tissue from mock, C227 or Δstx2 infected rabbits not treated with biotin, but incubated with streptavidin-Alexa 546.
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