Buffered glutaraldehyde

Samples were fixed in 3% v/v buffered glutaraldehyde (pH 7.4, Merck, Darmstadt, Germany), pre-embedded in 1.5% w/v agarose (Invitrogen, Thermo Fisher Scientific), washed three times in 0.1 M phosphate buffer (Soerensen, pH 7.4) afterwards and post-fixed in 1% v/v osmium tetroxide (Electron Microscopy Sciences, Hatfield, USA) for 2 h at room temperature. Dehydration was performed in a series of graded ethanol solutions (70%, 80%, 96% and 100%), subsequently infiltrated with propylene oxide (Sigma-Aldrich), followed by increasing ratios of epoxy resin-propylene oxide and finally pure resin (Serva, Mannheim, Germany). After an additional change, the resin was polymerized at 60 °C for 48 h. Semi-thin sections were cut at 0.8 μm and stained with toluidine blue, ultra-thin sections were cut at 70 nm, mounted on copper grids (Gröpl, Tulln, Austria) and stained with uranyl acetate (Fluka Chemie AG, Buchs, CH) and lead citrate (Merck, Darmstadt, Germany). Transmission electron micrographs were made with an EM900 (Zeiss, Oberkochen, Germany).

Samples of healthy (n = 3) and pyometra affected canine endometria (n = 3) were fixed in 3% buffered glutaraldehyde (pH 7.4, Merck). After washing in 0.1 M phosphate buffer (Soerensen, pH 7.4) samples were postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) for 2 hours at room temperature. Afterwards, samples were embedded in epoxy-resin (Serva). Semi-thin sections were cut at 0.8 μm, stained with toluidine blue (Merck) for general histological evaluation. Ultrathin sections were prepared (70 nm) and contrasted with uranyl acetate (Fluka Chemie AG, Buchs, Switzerland) and lead citrate (Merck), and evaluated by a transmission electron microscope (Zeiss EM900, Zeiss, Oberkochen, Germany). Images from surface and glandular epithelium were made to assess the amount of LDs (LDs/nuclei) and to measure LDs diameter (ImageSP-TRS, Moorenweis, Germany).

To further analyse polarity reversal of organoids, basal‐out and apical‐out organoids at day three after induction of polarity reversal were used. All samples were fixed in 3% buffered glutaraldehyde (pH 7.4, Merck). Organoids were then pre‐embedded in 1.5% agarose. After being washed in 0.1 M Soerensen buffer (pH 7.4), the samples were postfixed for 2 h at room temperature in 1% osmium tetroxide (Electron Microscopy Sciences). This was followed by dehydration in an ethanol series along with an increasing series of propylene oxide (Sigma‐Aldrich) before embedding and polymerisation in epoxy resin (Serva) for 48 h at 60°C. Ultrathin sections (70 nm) were cut for transmission electron microscopic evaluation and contrasted in methanolic uranyl acetate (Fluka Chemie AG) and alkaline lead citrate (Merck). For imaging, a transmission electron microscope (EM 900, Zeiss) equipped with a slow‐scan CCD camera (2k Wide‐angle Dual Speed, TRS) and ImageSP Professional software (SYSPROG, TRS) were used.