Tissue tek optimum cutting temperature compound

The entire length of the mLN chain was carefully dissected, weighed, imaged, and embedded in Tissue-Tek optimum-cutting temperature compound (Thermo Scientific), then frozen in an iso-pentane dry ice bath. Serial cryostat sections (8 μm in thickness) were collected over a span of 400 μm depth on Superfrost/Plus glass slides (Fisher Scientific), air-dried and fixed for 10–15 min in ice-cold acetone. Air-dried cryosections were then rehydrated in PBS and were blocked with 1% (wt/vol) BSA supplemented with normal mouse (1%) and donkey serum (4%). Indirect immunofluorescence staining was performed using various antibodies (listed in Supplementary table 2) diluted in PBS containing 1% (wt/vol) BSA and 1% (vol/vol) normal mouse serum. Cryosections were incubated with primary antibodies overnight at 4 °C. After overnight incubation cryosections were washed three times in PBS and primary antibodies were detected by incubating sections with fluorescently labeled secondary antibodies, and nuclei counter-stained with DAPI prior to mounting of the sections using ProLong anti-fade reagents (Life technologies). Stained cryosections were then imaged after 24 h. A detailed list of antibodies used is provided in Supplementary table 2.