Dual Staining Technique for FISH and Lectin

Dual staining for FISH and lectin was carried out as previously described [30 (link),31 (link)]. Briefly, deparaffinized sections were incubated at 37 °C overnight with Alexa Fluor 488-conjugated gamma-Proteobacteria Phylum GAM42a (5′-GCC TTC CCA CAT CGT TT-3′), which recognizes bacteria belonging to the γ-Proteobacteria class, in hybridization buffer (20 mM Tris-HCl, pH 7.4, 0.9 M NaCl, 0.1% sodium dodecyl sulfate). Sections were rinsed with wash buffer (20 mM Tris-HCl, pH 7.4, 0.9 M NaCl) and PBS twice each, and were co-labeled with fluorochrome-conjugated lectin (Ulex europaeus agglutinin I, UEA1, DyLight^® 649, DL-1068, Vector Laboratories) then mounted with VECTASHIELD. The fluorescence intensity of lectin was quantified using ImageJ software and represented as the integrated fluorescence intensity of the lectin relative to total DAPI values per tissue section. Slides were stained under the same conditions and at the same time.