Plan apochromat 40 1.3 oil dic objective

Transformants, 3‐days old, were used to inoculate 5 mL selective SC‐medium and grown at 30°C for 22 h. All images were generated using 100 ms exposure time at a Carl Zeiss axiovert 200M wide‐field fluorescence microscope fitted with a plan‐apochromat 40×/1.3 oil DIC objective. Both bright field and fluorescence images were collected. YFP was excited using 508 nm, with the light being filtered through a 500/25 band pass filter while the emitted fluorescence was filtered through a 535/30 BP filter. Images were collected using Zen blue software (Zeiss) and cell counting was performed using the ImageJ (NIH) software. For each sample, a minimum of 300 cells were counted and the fraction of fluorescent cells was calculated. To avoid bias all images were randomized and counted twice by two independent persons.

Distal colon with fecal material from C57BL/6 (WT mice) and Muc2^−/− mice was dissected and fixed in water-free Carnoy’s fixative (60% dry methanol, 30% chloroform, 10% glacial acetic acid). Paraffin embedded sections were dewaxed and hybridized with 10 ng/µl of a general bacterial 16SrRNA probe (EUB 338) conjugated to Alexa 555 and DNA was stained with DAPI [1] . Human biopsies from control and active UC were taken during colonoscopy. The Carnoy fixed and paraffin embedded sections were immunostained for Muc2 using the MUC2C3 antisera and DAPI to visualize the nucleus and bacteria as not all mucus-associated bacteria in human samples can be stained by the general EUB338 probe [8] . Images were obtained using a fluorescence microscope Eclipse E1000 with a Plan-Fluor 40x/0.75 DIC objective (Nikon) or a confocal microscope Axio Examiner Z1 LSM 700 with a plan apochromat 40×/1.3 oil DIC objective and the ZEN 2010 software (Zeiss, Germany).