Cryo-frozen intestinal cross sections from patient colonic biopsies were fixed with 4% paraformaldehyde and stained using the TSA plus kit [Perkin Elmer] according to the manufacturer’s instructions. Alternatively, slides were fixed with methanol at −20°C for 10 min, blocked with 10% fetal calf serum [FCS]/1% bvine serum albumin for 1 h and incubated overnight at 4°C with the primary antibody. Paraffin-embedded tissues were deparaffinized and antigen unmasking was performed using citrate buffer. Thereafter slides were incubated with different antibodies.
For DSS-induced colitis mice, immunofluorescence analysis was performed in intestinal sections, which were taken at the end of the experiment on day 10 for IL10 [Abcam], IL17 [Abcam] and Foxp3 [eBioscience]. Colon biopsies of UC patients were taken before and 28 days after topical cobitolimod or placebo treatment21 (link),22 (link) and the corresponding cross sections were stained for IL10 [Abcam], Foxp3 [eBioscience] or IL17 [Abcam]. From each sample, four to six high power fields [HPFs] per patient were analysed using a 10× objective magnification. Analysis of images was done with a fluorescence microscope [BZ-8100 or BZ-9000, Keyence] or a confocal microscope [LSM, Leica Microsystems] and calculated with ImageJ 1.52a [NIH]
For DSS-induced colitis mice, immunofluorescence analysis was performed in intestinal sections, which were taken at the end of the experiment on day 10 for IL10 [Abcam], IL17 [Abcam] and Foxp3 [eBioscience]. Colon biopsies of UC patients were taken before and 28 days after topical cobitolimod or placebo treatment21 (link),22 (link) and the corresponding cross sections were stained for IL10 [Abcam], Foxp3 [eBioscience] or IL17 [Abcam]. From each sample, four to six high power fields [HPFs] per patient were analysed using a 10× objective magnification. Analysis of images was done with a fluorescence microscope [BZ-8100 or BZ-9000, Keyence] or a confocal microscope [LSM, Leica Microsystems] and calculated with ImageJ 1.52a [NIH]
