All in vitro haptotaxis assays were recorded with a 203/0.5 PH1 air objective on an inverted wide-field Nikon Eclipse microscope equipped with a light source with flexible excitation band selection (green 549/15, red 632/22; Lumencor). Confocal microscopy images were obtained with a 103/0.3 PH1 objective on a Leica SP5 upright laser-scanning confocal microscope equipped with 488, 561 and 633 nm laser lines and with a 203/0.8 air objective on an inverted Zeiss LSM 700 with 405, 488 and 640 nm laser lines. For mCCL21 24-98 bio quantification, images were obtained using a 203/0.8 air objective on a Zeiss Axio Observer microscope equipped with an external light source (Leica). B4F patterns were written using a 403/1.2 W Korr UV-Vis-IR water immersion objective on an inverted Zeiss Observer microscope equipped with 354-nm pulsed laser and a motorized piezo stage.
Antibody Staining for Immobilized mCCL21 24-98 bio Printed B4F patches were stained with SA-Cy3 for 20 min at room temperature in the dark. After washing with PBS, patches were incubated with PBS only or with mCCL21 24-98 bio in PBS (250 ng/mL). Subsequently, patches were stained with goat anti-mouse CCL21 (R&D Systems) and rabbit anti-goat 488 (Molecular Probes), goat anti-mouse CCL21 (R&D Systems) only, or rabbit anti-goat 488 (Molecular Probes) only. Patches were imaged using a Zeiss Axio Observer wide-field microscope.
Antibody Staining for Immobilized mCCL21 24-98 bio Printed B4F patches were stained with SA-Cy3 for 20 min at room temperature in the dark. After washing with PBS, patches were incubated with PBS only or with mCCL21 24-98 bio in PBS (250 ng/mL). Subsequently, patches were stained with goat anti-mouse CCL21 (R&D Systems) and rabbit anti-goat 488 (Molecular Probes), goat anti-mouse CCL21 (R&D Systems) only, or rabbit anti-goat 488 (Molecular Probes) only. Patches were imaged using a Zeiss Axio Observer wide-field microscope.