MHC plays a role in establishing the pre-TCR immunological synapse at DN3 stage in situ. (A) DN3 cells were identified in five randomly picked regions of the same size in an intact thymus to assess their localization in relationship to MHCII. Images were acquired using widefield fluorescent microscopy (Vectra 3 automated quantitative pathology imaging system) and are representative of DN3 cells adjacent (yellow arrowheads) or away (white arrowheads) from MHCII. DN3 cells were then scored as either adjacent to MHCII or away from it, as shown by the column-bar plot. The total number of scored cells is 322, with 161 per biological replicate. (B) pTα clustering was assessed in DN3 cells adjacent to or away from MHCII, as shown in the stacked column-bar plot. DN3 cells adjacent to MHCII that showed pTα clustering were assessed for the localization of pTα clustering as compared with MHCII, as shown by column-bar plot. Images were acquired using widefield fluorescent microscopy (Vectra 3 automated quantitative pathology imaging system), and representative images of DN3 cells adjacent to MHCII with clustering of pTα toward MHCII (I), clustering of pTα away from MHCII (II), and with no clustering of pTα (III) are shown. Scale bars represent 50 µm (A) and 10 µm (B).