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.