Type 9

The enteroids were prepared for hematoxylin and eosin (HE) staining as previously described for human colonoids [22 (link)]. Briefly, the enteroids were fixated for 30 min in 4% paraformaldehyde. To increase the number of enteroids on each cross-section, multiple enteroid domes were dissociated by gentle pipetting and pooled into a 50 mL tube. The enteroids were pelleted by centrifugation for 5 min at 250 × g and suspended in pre-warmed 2% agarose (Type IX, Sigma-Aldrich). To accumulate the enteroids onto a single plane, the tube was immediately centrifuged for 3 min at 150 × g. The agarose was solidified on ice, embedded into paraffin wax and sectioned. This treatment allowed staining for histological examination (Additional file 2).

The central portion of the middle leaf zone bearing short tentacles at the surface was dissected and sandwiched as quickly as possible (20 s) in a mold between two lecithin-coated gold sample holders (Balzers BB1131242-1). For mechanical protection as well as for maximum heat conductivity, we filled the remaining space within the sample holder with a droplet of 1% ultralow gelling (< 15 °C) agarose (Type IX, Sigma Chemical Co.). Freezing of the tissue was accomplished with the Balzers HPM 010 high-pressure freezer.
Freeze substitution followed the procedure described by Lancelle et al. (1986 (link), 1987 (link)) and employed by Lichtscheidl et al. (1990 (link)). It thus resembled the method applied by Kiss et al. (1990 (link)) and Staehelin et al. (1990 (link)). Briefly, cryoimmobilized material was transferred to acetone containing 2% osmium tetroxide at approximately − 80 °C and freeze substituted for 36–40 h, then brought to room temperature gradually over a period of 5–6 h. Before embedding in a mixture of Epon and Araldite, the tissue was transferred to methanol and stained in 5% uranyl acetate in methanol for 2 h, then brought back to acetone.
After staining with lead citrate, ultra-thin sections were observed in a Jeol 100 CX or a Zeiss 902 electron microscope operated at 80 kV.