Peqgold rnapure reagent

Leaf samples were harvested after infiltration of leaves with Psph or with the control, the infiltrated areas with MgCl 2 . Total RNA was extracted with PEQ Gold RNAPure ® reagent (PEQ LAB, Erlangen, Germany) according to the manufacturer's instructions. The RNA obtained was separated on 1.5% agarose gels containing 5.5% formaldehyde, and blotted into nylon membranes. The nucleic acids bound to the membrane were cross-linked by UV-irradiation for 2 min. The expression of Pathogenesis related protein 1 (PR1) provides a marker for NO-elicited production of the defence hormone salicylic acid (Mur et al., 2000 (Mur et al., , 2013b)) . PR1 probes were labelled to high specific activity using the Random Primer DNA labelling system with 40 µCi [α-32 P] dCTP (ICN Biomedicals, Eschwege, Germany) according to the supplier's instructions. Hybridisation was performed at 65 °C for 15 h in 1% BSA, 1 mm EDTA, and 0.5 m Na 2 HPO 4 . The membrane was washed with 2× saline sodium citrate (SSC), 0.1% SDS at 65 °C for 30 min, and afterwards with 0.2× SSC, 0.1% SDS for 15 min at 65 °C prior to autoradiography. The air-dried membrane was exposed to Kodak X-omat DS film (Stuttgart, Germany) with an intensifying screen at -80 °C and the film was developed after appropriate time intervals.

Small intestinal adenomas were isolated from 120 days old APC min mice. The tissue was cut into small pieces and digested with Collagenase C and DNase. For further disruption, it was pulled through 18G and 22G needles. Cells were collected through a 70 µm cell strainer and incubated in serum-containing buffer to avoid unspecific antibody binding. In the next step, cells were labeled with biotinylated anti-F4/80 antibody (BioLegend) before magnetic Streptavidin MicroBeads (Miltenyi, Bergisch Gladbach, Germany) were added. Thereafter, F4/80-positive cells were selected by magnetic separation using LS columns (Miltenyi, Bergisch Gladbach, Germany) and finally dissolved in PeqGold RNA Pure reagent (Peqlab, Erlangen, Germany) for RNA isolation.