Lypd8

mTECs were isolated, analyzed, and sorted as previously described (Michelson et al., 2022a (link)). Thymi were finely chopped, the lymphocyte-rich supernatant was removed, and the thymic pieces were incubated at 37°C in DMEM supplemented with 2% FCS, 25 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Lonza), 0.5 mg/ml collagenase (Sigma-Aldrich), and 0.1 mg/ml DNaseI (Sigma-Aldrich) for 15 min, then in the same medium with 0.5 mg/ml collagenase/dispase (Roche) for 15 min. To dissociate cell–cell interactions, 10 mM EDTA was added. To prepurify mTECs, cells were incubated with anti-CD45 microbeads (Miltenyi) for 15 min and then CD45⁺ cells were depleted using MACS LS columns (Miltenyi). Cells were then stained with antibodies against EpCAM, CD45, Ly51, A/E, Lypd8 (all Biolegend), and/or GP2 (MBL). DAPI (Sigma-Aldrich) and Fixable Yellow Live/Dead (Invitrogen) were used for dead cell exclusion. mTECs were defined as live CD45⁻ EpCAM⁺ Ly51⁻ cells and further gated as mTEC^hi or mTEC^lo based on MHCII levels. Flow cytometry was performed on LSRII or FACSymphony A1 instruments (BD), and cell sorting was performed on a FACSAria cell sorter (BD). Flow cytometry data were analyzed with Flowjo (BD).

Imaging of thymic sections was performed as previously described (Michelson et al., 2022a (link)). Briefly, thymi were fixed for 1 h at 4°C in 4% paraformaldehyde (PFA) in PBS and then dehydrated in a 5–30% sucrose gradient overnight. Thymi were embedded in OCT, cut into 8-μm sections, permeabilized and blocked with 5% normal donkey serum in PBS plus 0.05% Tween-20 (PBS-T), stained for 1 h at room temperature (RT) with primary antibodies, washed, stained for 1 h at RT with secondary antibodies, washed, counterstained for nuclei, and mounted for imaging. Primary antibodies used were anti-EpCAM, -Lypd8 (both Biolegend), -GP2 (MBL), -Hnf4α (Abcam), and -Hnf4γ (Proteintech). FITC-, Cy3-, or Cy5-conjugated donkey anti-rat and anti-rabbit secondary antibodies, all from Jackson ImmunoResearch, were used as appropriate. Hoescht 33342 (Sigma-Aldrich) was used as a nuclear counterstain. Images were acquired by widefield microscopy using a Nikon Ti inverted microscope; Plan Apo 10× air, 20× air, or 60× oil objectives; Andor Zyla 4.2 Plus sCMOS camera; and Nikon Elements acquisition software, or by spinning-disk confocal microscopy with the same setup, plus a W1 Yokogawa spinning disk with 50-μm pinholes across multiple z-planes. Images were analyzed in ImageJ.