Opaque adhesivecaps

12 µm thick cryosections were prepared from flash frozen colonic tissue samples collected prior to culturing as well as after loss of the epithelial layer (LEL-M 5 h). After transfer to NF 1.0 PEN membrane coated slides (Carl Zeiss MicroImaging, Munich, Germany) sections were treated with DTT (1 mg/ml) for 5 min and stored in 100% EtOH at −20°C. Immediately before laser-capture microdissection, sections were stained with Cresyl Violet (0.1% in 100% EtOH; Sigma) and subsequently dehydrated by immersion in 100% EtOH and Xylene (Roth, Karlsruhe, Germany). LMD was performed for 1 h each using the PALM MicroBeam (Carl Zeiss MicroImaging). Microdissected tissue was collected using opaque AdhesiveCaps (Carl Zeiss MicroImaging).

Cryosectioning and LCM were performed as described previously 10. Briefly, 8 μm tissue sections were cut and melted on an ultraviolet (UV)‐treated polyethylene naphthalate slide (Carl Zeiss MicroImaging, Munich, Germany). Sections were fixed in ice‐cold 70% v/v ethanol/Milli‐Q water, dehydrated in 100% ethanol and stored at –80°C. Prior to LCM, sections were rehydrated in tap water, stained with hematoxylin, and dehydrated in increasing concentrations of ethanol in Milli‐Q water. LCM was performed using a type P‐MB device (Carl Zeiss MicroImaging) and completed within 1.5 h after staining. From each slide, an area of ∼500 000 μm² was collected in opaque adhesive caps (Carl Zeiss MicroImaging), equivalent to ∼4000 epithelial cells (assuming 10 μm³ per cell 11). Collected cells were dissolved in 20 μL of 0.1% w/v RapiGest in 50 mM ammonium bicarbonate and stored at –80°C until further processing.