3D immunohistology

CD44^low T cells from Wnk1^fl/+RCE and Wnk1^fl/-RCE chimeric mice were labeled with CMAC or CMTMR for 15-20 min at 37°C, with dyes swapped between experiments. 3-4 x 10⁶ cells were washed and injected intravenously at a 1:1 ratio into 5-10 week old C57BL/6J recipient mice. After 20 min, further adhesion of T cells to HEV was prevented by intravenous injection of anti-CD62L (Mel-14, 100 μg/mouse; Nanotools) in combination with Alexa Fluor 633-conjugated anti-PNAd (MECA-79, BD Biosciences, 15 µg/mouse) for visualization of HEVs by staining of PNAd. After a further 20 min (40 min after transfer) mice were sacrificed and perfused using 10 ml cold PBS and 10 ml cold 4% PFA. PLNs were processed as described³. Images were visualized with Volocity software and T cells were counted manually and differentiated between cells in the lumen of HEV (“luminal”), attached to the outer MECA-79 signal (“perivascular”) and cells in the parenchyma of the peripheral lymph node (“parenchymal”). Alternatively mice were analyzed 20 min after transfer of T cells without injection of Mel-14.