Roti histofix

Dissected heart tissue was fixed in 4% paraformaldehyde in phosphate-buffered saline (Roti-Histofix, Carl Roth, Karlsruhe, Germany) over 24 hours and embedded in paraffine after dehydration. Paraffin blocks were cut into 5 μm slices and stained after rehydration with the Picrosirius-red method (Sigma-Aldrich, Taufkirchen, Germany) or fluorophore-conjugated wheat germ agglutinin (WGA-Alexa Fluor 488 conjugate, Life Technologies, Karlsruhe, Germany). Picrosirius-red stained ventricular sections served for determination of left ventricular interstitial fibrotic area using AxioVision software (Carl Zeiss, Oberkochen, Germany). WGA-stained sections were used for quantification of cardiomyocyte cross-sectional areas after nuclear counterstaining with DAPI (4’,6 diamidino-2-phenylindole, Life Technologies, Karlsruhe, Germany). In each ventricle, ≥ 50 cardiomyocytes were measured using the AxioVision software. Measurements were limited to cardiomyocytes that were cut perpendicular to their long axis at the level of a centered, round cardiomyocyte nucleus.

The fifth segment of eight equally sized segments of the small intestine was flushed with cold PBS and fixed in Roti^®-Histofix (#P087, Roth) at 4 °C overnight. Tissue was processed for paraffin embedding at the Core Facility Histology at University Medical Center Mainz. Tissue sections were cut at 3 µm thickness, dewaxed and heat-induced epitope-retrieval was done using citrate buffer (10 mM sodium citrate, pH 6.6). Unspecific binding was blocked using normal goat serum (#S-1000, Vector Laboratories, 5% v/v in PBS) and sections were incubated for 1 h at room temperature with rabbit anti mouse-Ki-67 antibody (1:500 in blocking solution, #IHC-00375, Bethyl Laboratories Inc.). After washing, sections were incubated with the biotinylated anti-rabbit antibody (#BA-1000, Vector Laboratories) and signal detection was done using Vectastain^® Elite^® ABC HRP (#PK-7100, Vector Laboratories) and 3,3’-diaminobenzidine (DAB) as substrate according to the manufacturer’s protocol. Sections were dehydrated and mounted using Eukitt^® mounting medium (#SIAM03989, VWR). Ki67-positive cells as well as total number of cells per villus/crypt were counted, averaged and represented as percent.

Hydrogels were fixed for 3 h in 4% paraformaldehyde (ROTI^®Histofix, Carl Roth, Karlsruhe, Germany) and cut in half. Cells within the hydrogel were permeabilized for 30 min (0.1% Triton X-100 Sigma Aldrich T8787 in PBS-), blocked for 30 min, and washed three times for 15 min in washing buffer (0.1% Tween20 in PBS-). Staining was performed with 1:500 rabbit anti-human perilipin A antibody (Sigma Aldrich, P1998) for 1 h and afterward with 1:500 goat anti-rabbit IgG-Alexa488 antibody (ab60314, abcam, Cambridge, U.K.) for 30 min. Hydrogels were washed and stained with 4′,6-Diamidin-2-phenylindol (DAPI, Serva) for 10 min and washed again.

Tumor cells were transplanted to the chorioallantoic membrane (CAM) of fertilized eggs from genetically identical hybrid LB chicks, followed by treatment and humane euthanasia of the chick embryo as described [26 (link), 48 (link)]. After resection, the tumor take rates were calculated from the percent of transplanted tumors that engrafted and the tumor volumes according to the formula Volume = 4/3 × π × r³ (r = 1/2 × square root of diameter 1 × diameter 2) [49 (link)]. Tumor tissue was fixed in Roti Histofix (Carl Roth, Karlsruhe, Germany) for 2-3 days and embedded in paraffin for further analysis.

For the heterotopic CRC tumor engraftment model, 1 × 10⁵ CT26 cells were injected subcutaneously (s.c.) into the flank of 8–12 week old BALB/c or ΔdblGATA-1 mice. When tumors were palpable (after ~1 week), mice were treated intraperitoneally (i.p.) with 0.4 µg recombinant mouse (rm)IL-33 (Biolegend; # 580506) or PBS (as a negative control) every other day six times in total.^{25 (link)} Tumor progression was monitored during the course of the experiment. Mice were sacrificed 24 hrs after the last injection, and tumors were collected. Tumors were weighed, and then measured with a caliper. The tumor volume was calculated by using the formula (v = length x width x height x π/6).
Colitis-associated CRC was induced in CD-1 mice as described before.^{33 (link)} In brief, mice were first injected i.p. with 10 mg/kg of azoxymethane (AOM; Sigma-Aldrich; A5486). 2% of dextran sulfate sodium (DSS; MP Biomedicals; # 216011090) was then added to their drinking water from day 7–13 and 28–34. IL-33 treatment was started seven days after the last bout of DSS and applied i.p. at 1 µg/mouse two to four times per week. On day 99, mice were sacrificed, the colon was removed and opened longitudinally. Tumors were counted and tumor areas were measured with a caliper. Tumors were excised and kept in RPMI on ice until further use (or fixed in 10% Roti®-Histofix; Carl Roth; A146.5).