3D Immunohistology

After intravital imaging, to preserve integrity of the tissue, the lymph nodes were fixed by intracardiac perfusion using a solution containing 4% formaldehyde in PBS as previously described (23). After fixation, lymph nodes were collected and prepared for the tissue clearing process: after 1 h of incubation in a solution containing 50 mM of NH₄Cl, and 5% of FBS, lymph nodes were rinsed in PBS three times for 5 min and then incubated overnight in PBS + 1% triton X-100. After three rinses in PBS, LN were incubated for 6–10 h in FocusClear (CelExplorer), a tissue clearing solution. LN were then mounted on a coverslip and imaged with the same parameters previously used for intravital imaging.