High precision glass slides

Home-built sample chambers consisted of a pair of plasma cleaned (Diener Electronic, Ebhausen, Germany) high precision glass slides (170 ± 5 μm, No. 1.5 H, Marienfeld Superior, Lauda-Königshofen, Germany) fixed together by double-sided tape forming a channel. 50 pM of ribosomes were added and thoroughly washed with Tico buffer.

For immunofluorescence, 1.5 × 10⁶ PBMCs/well were cultured in 500 µl/well of osteoclast medium with supplements on 13-mm diameter high-precision glass slides (Marienfeld) in 24-well plates at 37°C and 5.5% CO₂ until fully differentiated. The medium was changed every 2 days. The cells were stimulated as previously mentioned and then fixed and stained with anti-IFNγR (CD119) and anti-FcγR3 (CD16) antibodies overnight at 4°C. Next, the cells were stained with the secondary antibodies anti-mouse AF488 and anti-rabbit AF568 (Invitrogen, Thermo Fisher Scientific Dreieich, Germany) for 1 h at room temperature. DAPI was used as counterstaining, and cells were embedded in Mowiol for microscopy analyses.
The recorded total internal reflection fluorescence (TIRF) images were analyzed using a custom ImageJ/Fiji plugin. Both AF488 and AF568 channels were processed using a difference-of-Gaussian filter, and IFNγ (AF488) and Fcγ receptors (AF568) were detected as local maxima above a threshold in the filtered images. Distances between the two receptor types were then calculated for each detected IFNγ receptor and the closest located Fcγ receptor.