Eub338

Hybridization of distal large intestine slides with bacterial probes was completed as described previously.^{84 (link)} Briefly, unstained slides were deparaffinized in 3 washes with xylene followed by 3 washes in ethanol. Slides were dried in an incubator at 50°C for 25 minutes. Slides were incubated overnight at 50°C with 1 ng/µL of probe targeting all bacteria (EUB338: Alexa 555 5’-GCTGCCTCCCGTAGGAGT −3’) (Invitrogen) in buffer (0.9 M NaCl, 20mMTris-HCL, pH 7.5, 0.1% SDS). To wash unbound probe, the slides were incubated 15 minutes in buffer (0.9 M NaCl, 20mMTris-HCL, pH 7.5, 0.1% SDS) three times. Slides were then air-dried, mounted, and counterstained with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Slides were imaged on a Zeiss Axiovert 200 m fluorescent microscope at total magnification of 400x and processed by the Axiovision software. In Adobe photoshop, a histogram stretch was employed for the blue and red channels to spread the image intensities across the entire intensity display range.

Fluorescence in situ hybridization was performed as previously described in detail [24 (link)]. Briefly, the tissue sections were directly labeled with the Cyanine 3-conjugated universal bacterial probe EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′, Invitrogen). After probing and hybridization, the slices were restained with 4′,6-diamidino-2-phenylindole. Coimmunostaining with anti-mucin 2 (MUC2) (ab272692, Abcam) was performed.