2.5. Pre-Embedding Immunogold Labeling and Processing for TEM

In order to analyze the distribution of the cell surface receptor for hyaluronic acid (CD44) and chondroitin sulfate (CS56) in the GCX of THP1-cells, we applied pre-embedding immunogold labeling for TEM analysis. For a list of the primary antibodies, refer to Table 1. Rehydrated cells were washed twice with phosphate buffer (PB). To avoid nonspecific binding, samples were incubated for 30 min in blocking solution containing PB, 5% NGS (PAN Biotech), and 2% BSA (Sigma-Aldrich, Darmstadt, Germany). All of the following immuno-incubations were done with gentle agitation, overnight at 4 °C. After each incubation step, cells were gently centrifuged. After blocking, suspension cells were incubated with primary antibodies: CD44 and CS, both diluted in blocking solution. After washing with PB, cells were incubated with secondary gold conjugated antibodies: goat anti-rat IgG 30 nm Gold (BBInternational, Cardiff, UK) and goat anti-mouse IgM 25 nm Gold (Aurion, Wageningen, The Netherlands), both diluted 1:20 in PB containing 0.2% acetylated BSA (BSA-c.; Aurion, Wageningen, The Netherlands). To remove unbound secondary antibodies, cells were washed thoroughly with PB/BSA-c and then with PBS. Then, samples were post-fixed with 2% GA in PB for 30 min to crosslink gold conjugates in the structures, which prevents the loss of labeling during subsequent processing. Next, cells were washed several times in PB and incubated additional 30 min with buffered 0.5% OsO₄ for structural stabilization. Next, cells were washed in PB and in ddH₂O, dehydrated in increasing concentrations of ethanol and finally embedded in epoxy embedding medium (Epon 812; Sigma-Aldrich, Darmstadt, Germany). After resin polymerization at 60 °C, cells were ultra-sectioned into 60–65 nm on an ultramicrotome (Reichert Ultracut S, Leica, Germany) with a diamond knife (Diatome, Nidau, Switzerland) and mounted on 300-mesh Formvar-coated nickel grids (Plano, Wetzlar, Germany). Ultrathin sections were examined, without further staining, using a Zeiss transmission electron microscope 912 (TEM-912, Carl Zeiss, Oberkochen, Germany) operating at 120 kV and equipped with a digital camera (Proscan 2K Slow-Scan CCD-Camera, Carl Zeiss, Oberkochen, Germany). Digital image acquisition was performed using the ImageSP software (TRS, Moorenweis, Germany).