Supplementary Materials and Methods
Immunohistochemistry for apoptosis and neutrophil influx
After deparaffinization and rehydration of paraffin-embedded sections (4μm), endogenous
peroxidase activity was blocked using 0.6% hydrogen peroxide in methanol for 15 minutes.
Sections undergoing heat-mediated antigen retrieval (M30 staining) were heated in 10 mM
citrate buffer (pH 6.0) for 20 minutes at 90ºC. After blocking non-specific antibody binding using
5% bovine serum albumin (BSA) in PBS, sections were incubated with specific primary antibody
at room temperature for 60 minutes. The following primary antibodies were used: mouse anticleaved cytokeratin 18 antibody, clone M30 (Peviva, Bromma, Sweden) to visualize apoptosis,
and rabbit anti-human myeloperoxidase (MPO staining, DakoCytomation, Glostrup, Denmark) to
visualize neutrophil influx. After washing, appropriate peroxidase-conjugated or biotinconjugated secondary antibodies were used, the latter followed by incubation with the avidinbiotin system (Vector Laboratories, Burlingame, CA). Binding of primary antibody was visualized
with 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MI). Sections were counterstained with
hematoxylin. Stained sections were photographed using a Nikon eclipse E800 microscope
equipped with a Nikon digital camera DXM1200F. For quantification, M30 and MPO-positive
cells were counted by two independent observers in five representative microscopic fields
(magnification colon: 100x, magnification small intestine: 200x) per time point. Interobserver
variability was not statistically significant. Numbers of positive cells were corrected for tissue
surface.
Immunofluorescence for Zonula Occludens-1
Cryostat sections (4μm) were dried, fixed in 10% trichloric acid for 15 minutes and non-specific
antibody binding was blocked using 5% bovine serum albumin (colonic tissue) or 3% fetal calf
serum (small intestinal tissue). Next, slides were incubated with rabbit anti-human ZO-1 (Zymed
Laboratories Inc., San Francisco, CA). After washing, slides were incubated with a CY3-labelled
secondary antibody goat anti-rabbit IgG (Invitrogen, Eugene, OR), followed by incubation with
4',6-diamino-2-phenylindole dihydrochloride (DAPI). Next, slides were dehydrated in ascending
ethanol series, mounted in fluorescence mounting solution (Dakocytomation) and visualized
with an immunofluorescence microscope.
RNA isolation and quantitative PCR
Intestinal samples were crushed with a pestle and mortar in liquid nitrogen. Disruption and
homogenization of the tissue was achieved using an Ultra Turrax Homogenizer (IKA
Labortechnik, Staufen, Germany) in lysis buffer containing β-mercaptoethanol (Promega
Corporation, Madison, WI). RNeasy spin columns were used to bind RNA. Columns were
washed and RNA was eluted in RNase-free water. To analyze gene expression of tumor
necrosis factor alpha (TNF-) and interleukin-6 (IL6), quantitative PCR (qPCR) was performed.
RNA samples were treated with DNAse (Promega Corporation) to ensure removal of
contaminating
genomic
DNA.
RNA
quantity
was
measured
using
the
NanoDrop
spectrophotometer (Thermo Scientific, Wilmington, MA). Total cDNA was synthesized using the
iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). qPCR reactions were performed in a
volume of 20 µl containing 10 ng of cDNA, 1x Sensimix SYBR Green (Bioline, London, UK) and
300 nM of gene-specific forward and reverse primers. The sequence of primers is provided in
Table 1. cDNA was amplified using a three-step program (40 cycles of 10s at 95°C, 20s at 60°C
and 20s at 70°C) with a MyiQ system (Bio-Rad). Specificity of amplification was verified by melt
curve analysis. Gene expression levels were determined using iQ5 software and a ∆Ct relative
quantification model. The geometric mean of the expression of two internal control genes,
RPLP0 and CYPA, was calculated and used as a normalization factor.
Protein isolation and IL-6 assessment
To isolate protein, snap frozen intestinal tissue was homogenized in lysis buffer containing
200mM NaCl, 10 mM Tris buffer, 5 mM EDTA and 10% glycerol using a Biospec minibeadbeater (Bartlesville, OK). Samples were centrifuged at 27.000 g for 15 minutes at 4°C.
Protein concentration of supernatants was determined using a BCA Protein Assay Kit (Pierce,
Etten-leur, the Netherlands). Equal amounts of protein (5µg) were used in an in-house enzymelinked immunosorbent assay (ELISA) kit to asses IL-6 protein concentrations. In short, 96-well
plates (Greiner Bio-One, Kremsmunster, Austria) were coated overnight (4°C) with the
monoclonal antibody 5E1. Free sites were blocked with 1% bovine serum albumin in PBS.
Samples and standard dilution series were incubated for 2 hours. Human recombinant IL-6 was
used for standard titration curves. Washing buffer contained 0.1% Tween-20. Biotinylated rabbit
antihuman IL-6 was developed in our laboratory and used as detection antibody. Finally,
3,3’,5,5’-tetramethyl-benzidine (TMB) was used as a substrate and the reaction was stopped by
adding 1M H2SO4. Samples were analyzed spectrophotometrically (450 nm) using an
automated ELISA reader. The detection limit for the assay was 0.005 ng/mL. Samples were run
in duplicate and a variability of 5% between sample duplicates was accepted.