Biosystems embedding station

Snips of the proximal colon were fixed in Carnoy’s solution (60% methanol, 30% chloroform, 10% glacial acetic acid), incubated in two changes of dry methanol (Sigma-Aldrich, Dorset, UK) for 30 min each, followed by absolute ethanol (ThermoFisher Scientific, Paisley, UK) for two incubations at 30 min each. Finally, tissue cassettes were processed in a Micro-spin Tissue Processor STP120 (ThermoFisher Scientific) and immersed in paraffin. Colon snips were embedded in paraffin blocks using a Leica Biosystems embedding station (Leica Biosystems, Milton Keynes, UK), with the luminal surface of the colon exposed for tissue sectioning. 5 µm tissue sections were cut using a Leica Biosystems microtome and adhered to uncoated microscope slides (ThermoFisher Scientific). Slides were dried for 48 h at 50 °C before use. Histological analysis was used to determine that all five of the 18 week-old mdr1a^−/− mice had indications of moderate or mild colitis, with a loss of healthy gut architecture^{17 (link)}.

Carnoy's fixed samples were incubated in two changes of dry methanol (Sigma) for 30 min each, followed by absolute ethanol (ThermoFisher Scientific, Crawley, UK) for two incubations at 30 min each. Tissue cassettes were processed in a Micro‐spin Tissue Processor STP120 (ThermoFisher Scientific) and immersed in paraffin using a Leica Biosystems embedding station (Leica Biosystems, Milton Keynes, UK), with the luminal surface of the colon exposed for tissue sectioning. Tissue sections (5 μm) were cut using a Leica Biosystems microtome and adhered to uncoated microscope slides (ThermoFisher Scientific). Slides were dried for 48 hr at 50° before use. Haematoxylin & eosin and goblet cell staining were performed and analysed as described previously.7