Immunohistochemistry and Microscopy

2.2.4.1. Fixation and paraffinization of gastric tissue and mucosoid cultures

Tissue or filters with epithelial cells from mucosoid cultures were fixed overnight in 4 % paraformaldehyde (pFA) at 4°C and washed with PBS. Filters were further embedded orthogonally in Histogel (HG-4000-144) inside a casting mold. Tissue sample or Histogel block with embedded filter were paraffinized overnight in a Leica TP1020 tissue processor which performed automatic dehydration with the following steps: 70 % ethanol (2 h), 80 % ethanol (2 h), 90 % ethanol (2 h), 96 % ethanol (2 h), isopropanol (2 h), xylene (2x 2 h), and paraffin (2x 2 h). The paraffin blocks are generated inside a metal casting mold on a paraffin embedding console (Microm). From pre-cooled paraffin blocks, thin sections (5-10 µm) were cut with a paraffin rotation microtome (Microm) and transferred to glass slides.

2.2.4.2. De-paraffinization

For de-paraffinization of the sections, slides were incubated twice in xylene for 10 min respectively, twice in 100 % ethanol for 2 min respectively, followed by two minutes incubation steps in 95 % ethanol, 70 % ethanol, 50 % ethanol and twice in tap water.

Slides were transferred to a slide chamber with pre-heated antigen retrieval solution (Gibco) and incubated for 30 min in a 100°C water bath and 20 min at RT to cool down the slides. Slides were washed for 5 min under running tab water. Water was exchanged with PBS.

2.2.4.3. Hematoxylin & Eosin staining (H&E)

Hematoxylin and eosin staining are used to visualize the various structures of histologic sections. Hematoxylin stains nuclear DNA while with eosin cytoplasmic compartments

2.2 Methods

are stained. De-paraffinized sections of mucosoid cultures were covered with Mayer’s Hematoxylin solution (Roth) and incubated for 15 min at RT. The slides were rinsed shortly with ddH2O, followed by 5x 3 min rinsing under running tab water and finally 2 min rinsing with ddH2O. In the following, the sections were covered with Eosin Y solution (1 % aqueous, Roth) and incubated for 10 min at RT. Finally, the slides were rinsed for 1 min with ddH2O and embedded using Roti®-Histokitt.

2.2.4.4. Immunofluorescence labeling of paraffin sections

Immunofluorescence (IF) is a method to visualize proteins by labeling the target protein in two steps with a specific primary antibody and a fluorescence dye coupled secondary antibody. De-paraffinized sections were encircled with PapPen (biozol #CED-MU12) to prevent the running down of liquid. Sections were incubated for 60 min at RT in a humidified chamber with IF blocking solution to block unspecific binding sites and avoid unspecific background signals. Blocking solution was removed and primary antibody was added on the section and incubated for 90 min at RT for mucosoid cultures or overnight at 4°C for tissue sections in a humidified chamber. Slides with sections were washed 3x 5 min in PBS. Sections were incubated with immunofluorescence labeled secondary antibody, nuclei dye and cytoskeleton dye, light protected in a humidified chamber for 60 – 120 min at RT. Slides were washed in PBS (3x 5 min), followed by water once. Sections were embedded with mowiol and stored light protected at 4°C. Sections were imaged, using the Leica TSP8 confocal microscope.

2.2.4.5. Whole Mount Immunofluorescence

Whole Mount Immunofluorescence is a method in which part of the filter of mucosoid culture insert is used to visualize a target protein by fluorescence antibody labeling. In comparison to immunofluorescence staining of filter sections which gives a vertical view on the cell monolayer, the whole mount staining serves a top view on the cells and allows analyzing a greater amount of cells. For whole mount immunofluorescence staining, one-quarter of a mucosoid culture filter with epithelial cells is needed. The

2.2 Methods cooled methanol at -20°C. Filter was washed twice in PBS and unspecific binding sites of the cells on the filter were blocked by incubating the filter in 50 µL whole mount IF blocking solution for 60 min at RT in a humidified chamber. Afterward, filter was incubated with primary antibody (overnight, 4°C), washed five times in PBS and incubated light protected with fluorescence-labeled secondary antibody, nuclei dye, and cytoskeleton dye for 120 min at RT in a humidified chamber. Filter was embedded on a glass slide with mowiol and stored light protected. Samples were imaged at Leica TSP8 confocal microscope.

2.2.4.6. Confocal Microscopy and image analysis

After IF or H&E staining, images were recorded at the confocal microscope with a 10x, 20x or 40x oil objective using the Leica TCS SP-8 confocal microscope (Leica Microsystems GmbH) and the corresponding Leica software (Leica Microsystems GmbH). Afterward, images were further processed and analyzed using Fiji ImageJ (open source). This software was also used for cell and particle counting and measurement of cell height.

2.2.4.7. Transmission Electron Microscopy

Transmission electron microscopy (TEM) is a method serving for the fine structural analysis of cells. Mucosoid culture cells were fixed with 2.5 % glutaraldehyde (GA) in PBS and post-fixed with 0.5 % Osmium-tetroxide, contrasted with Uranyl-acetate and tannic acid, dehydrated in a graded ethanol series, and infiltrated in Polybed (Polysciences). Cut out pieces of the filters were stacked in flat embedding molds with Polybed. After polymerization, specimens were cut at 60 nm and contrasted with lead citrate. Specimens were analyzed in a Leo 906E transmission electron microscope (Zeiss, Oberkochen. DE) equipped with a side-mounted digital camera (Morada, SIS-Olympus, Münster, DE). The sample preparation after fixation, image aquistion, and analysis were performed by Dr. Christian Goosmann from the MPIIB core facility Microsopy.

2.2 Methods

2.2.4.8. Cryo-Immunogold Electron Microscopy

Mucosoid culture samples were fixed with 4 % pFA and 0.05 % GA in PBS and filter membrane was cut into 1 mm wide streaks. The filter membrane pieces were gelatin-embedded and infiltrated with 2.3 M sucrose according to the method described (P.J.Peters 2006). Ultrathin sections were cut at -95°C with an RMC MTX/CRX cryo-ultramicrotome (Boeckeler Instruments Inc., Tucson AZ, USA) transferred to carbon- and pioloform-coated EM-grids and blocked with 0.3 % BSA, 0.01 M glycine, 3 % CWFG in PBS. The sections were incubated with appropriate dilutions in the same buffer of rabbit polyclonal antibody directed against PGC (see Table 13) or mouse antibody directed against ATP4B (see Table 13). Secondary antibody-incubations were carried out with goat-anti-rabbit (or mouse) antibodies coupled to 12 nm gold particles (Jackson Immuno Research, West Grove, PA, USA). Specimens were then contrasted and embedded with uranyl-acetate/methyl-cellulose following the method described (Slot et al. 1991) and analyzed in a Leo 912AB transmission electron microscope (Zeiss, Oberkochen, Germany) at 120 kV acceleration voltage. Micrograph-mosaics were scanned using a bottom mount Cantega digital camera (SIS, Münster, Germany) with ImageSP software from TRS (Tröndle, Moorenweis, Germany). Annotation of gold particles was performed with Fiji Image J (Schindelin et al. 2012; Schneider, Rasband, and Eliceiri 2012) software using the ‘Gold Digger’ Macro (Sjollema and Giepmans Oct. 24, 2016). The sample preparation after fixation, image aquistion, and analysis were performed by Dr. Christian Goosmann from the MPIIB core facility Microsopy.