Meca 79

The fluorescent dyes 5(6 (link))-CFDA, SE (CFSE), (5-(and−6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine) (CMTMR, CellTracker orange) and Chloromethyl-coumarin (CMAC, CellTracker blue) were purchased from ThermoFisher (Basel, Switzerland) and the cell proliferation dye eFluor 670 was from eBioscience (San Diego, CA). mAb against PNAd (MECA-79) was from nanotools (Freiburg, Germany) and coupled to AlexaFluor-633 using a Protein Labeling Kit from Molecular Probes (Basel, Switzerland). Fc receptor blocking mAb (clone 2.4G2, 4.5 mg/mL) in FACS buffer (D-PBS supplemented with 1% milk powder and 0.1% NaN3) was produced in-house. T cells were fixed with 1% paraformaldehyde (PFA; Electron Microscopy Sciences, Lucerne, Switzerland) diluted in D-PBS. D-PBS supplemented with 10 mM EDTA was used for homogenizing LNs for flow cytometry experiments.

B cells were labeled with CMTMR (5 μM) and transferred i.v. into C57BL/6 recipient mice. After 20 min, further adhesion of B cells to HE) was blocked by i.v. injection of anti-CD62L (Mel-14, 100 µg/mouse; Nanotools) in combination with Alexa Fluor 633–conjugated anti-PNAd (MECA-79, 15 μg/mouse; Nanotools) to visualize HEV. After a further 20 min (40 min after B cell transfer), mice were sacrificed and perfused with 10-ml cold PBS and 10-ml cold 4% PFA. Popliteal, inguinal, axillary, and brachial LNs were harvested and cleaned from surrounding connective tissues. LNs were fixed overnight in 4% PFA at 4°C. The following day, LNs were embedded in 1.3% low gelling agarose (A-9414; Sigma-Aldrich), dehydrated in 100% methanol (Sigma-Aldrich) overnight, followed by clearing with a benzyl alcohol, benzyl benzoate solution (Sigma-Aldrich) as described (Boscacci et al., 2010 (link)). LNs were imaged by two-photon microscopy (2PM), taking 251–501 images from a 300–400 × 300–400 μm field of view with z-stacks at 2 μm spacing. Images were visualized with Imaris 9.1.2 software and cells attached to the luminal MECA-79 signal (“luminal”), attached to the abluminal MECA-79 signal (“perivascular”), and cells in the parenchyma of the popliteal, inguinal, axillary, and brachial LNs (“parenchymal”) were manually differentiated.