Axio observer z1 confocal microscope

50,000 GFP-tagged HS-cells and ΔHS-cells were collected; then seeded for 2 hours over a glass chamber slide (Thermo Fisher Scientific) pre-coated overnight with 1 μg ALCAM then fixed and permeabilized with Fixperm solution quenched with ammonium chloride, then incubated in blocking solution (PBS containing 0.01% Triton X-100 with 1% BSA). Finally, cells were immunolabeled with anti-phospho-SLP-76(pTyr128) followed by anti-mouse Alexa 647. Clustering images of SLP76 at the HS/ALCAM interface were captured using a Zeiss Axio-Observer Z1 confocal microscope equipped with a Yokogawa CSU10 spinning disc, a Zeiss 63x/1.43 NA objective, and a Hamamatsu Orca-AG camera. Single 0.1μm z-slices with SLP-76 puncta at the eGFP.G-expressing T-cell surface were detected and compared to ΔHS-cells.

The endothelium/HS-T-cells interface was imaged using confocal microscopy. HS-cells and NT T-cells were seeded over HBMEC monolayer, pre-stimulated with TGFβ (0.1 μg/ml) for 4 hours to ensure ALCAM expression, and incubated for 2 hours at 37°C to allow conjugate interactions. Conjugates between HS-cells and HBMEC were then fixed and permeabilized with 4% paraformaldehyde and 0.02 % saponin (Tween), blocked with PBS containing 0.01% Triton X-100 with 1% BSA and stained for LFA-1 open conformation, ICAM1, and Talin-1. Primary antibodies were subsequently detected by anti-mouse AF 647, AF 488 secondary antibodies and anti-rabbit Alexa fluor pacific blue respectively. An extra blocking step was performed between the two anti-mouse human antibodies staining to eliminate background staining. HS-cells/HBMEC conjugates were imaged as 0.2 μm Z-steps to cover the entire volume of the podosynapse, determined individually for each conjugate using a Zeiss Axio-Observer Z1 confocal microscope equipped with a Yokogawa CSU10 spinning disc, a Zeiss 63x/1.43 NA objective, and a Hamamatsu Orca-AG camera. Images were analyzed with Volocity software (PerkinElmer, Waltham, MA). Cluster density of LFA-1, Talin-1, ICAM1 and Actin at the interface were calculated using the formula (volume×MFI) for an equal number of 1×1×1-μm voxels selected to cover the interface.