Formalin-fixed paraffin-embedded colonic tissues were sectioned (6 μm) and analyzed via immunofluorescence and FISH. For FISH analysis, tissue sections were hybridized with 10 ng/mL of a universal bacterial 16S fluorescent rRNA probe, according to previously published protocol [99 (link)]. For immunofluorescence, tissue sections were incubated with anti-CR LPS (Biotech Laboratories, Ottawa, Canada), biotinylated goat anti-GFP (GeneTex, Irvine, USA), anti-MBD-3 (Santa Cruz Biotechnology, Dallas, USA), anti-TFF3 (Santa Cruz Biotechnology, Dallas, USA) overnight at 4°C.
Confluent Caco-2 monolayers were fixed with 100% methanol and permeabilized with 0.1% Triton 1% BSA solution in PBS. Primary antibodies were used to detect β defensin-2 (Santa Cruz Biotechnology, Dallas, USA) and TFF3 (Santa Cruz Biotechnology, Dallas, USA). The selectivity of these antibodies has been previously validated [61 (link), 100 (link)–102 (link)]. Caco2 cells are known to produce low levels of TFF3, and the use of Caco2 enterocytes to detect modulation of TFF3 release has been previously validated [103 (link)–105 (link)]. Images were acquired using a Nikon epifluorescent microscope, and ImageJ was used for microscopic image analysis.
Confluent Caco-2 monolayers were fixed with 100% methanol and permeabilized with 0.1% Triton 1% BSA solution in PBS. Primary antibodies were used to detect β defensin-2 (Santa Cruz Biotechnology, Dallas, USA) and TFF3 (Santa Cruz Biotechnology, Dallas, USA). The selectivity of these antibodies has been previously validated [61 (link), 100 (link)–102 (link)]. Caco2 cells are known to produce low levels of TFF3, and the use of Caco2 enterocytes to detect modulation of TFF3 release has been previously validated [103 (link)–105 (link)]. Images were acquired using a Nikon epifluorescent microscope, and ImageJ was used for microscopic image analysis.
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