Epcam clone g8

FACs analysis of fibroblasts was performed using a panel of typical CAF markers; podoplanin (clone 8.1.1), PDGFRα (clone APA5) and β (clone APB5) (all 1:300, all BioLegend), FAP-α (1:50, AF3715; R&D Systems); and markers to exclude immune cells (MHCII; clone KH74 and CD45; clone 30-F11), epithelial cells (EpCAM, clone G8.8; Biolegend) and endothelial cells (CD31, clone 390; BioLegend). Morphological characteristics assed and visualised using an EVOS microscope and functional analysis was measured in terms of collagen gel contraction capacity. 1.5 × 10⁵ cells were seeded into 2 mg ml⁻¹ collagen gel (BD Biosciences) and detached from the sides of 24 well plates. Gel contraction was imaged over time and quantified as percentage area change over time. CAFs displayed typical CAF morphology, marker profiles and functionality were utilised (Supplementary Fig. 1).

For thymocyte staining, single-thymocyte suspensions were prepared from the thymi of mice using a 70-μm cell strainer. Samples were then stained with specific fluorochrome-conjugated antibodies of cell surface CD markers, indicated in each figure legend, and then fixed and permeabilized with fixation/permeabilization buffer (eBioscience Treg staining kit, #88-8115-40), per company’s instruction, followed by intracellular staining for PE-anti-FoxP3 (eBioscience Cat# 12-5773-82) or FITC-anti-FoxP3 (eBioscience Cat# 11-5773-82), and/or PE-anti-p-Zap70 (Cell Signaling, Cat# 14791, clone 65E4, recognizing phosphorylation at Tyr319/Syk [Tyr352]), PE-anti-Nur77 (eBioscience, Cat# 12-5965-82, clone 12.14), and AlexaFluor488-anti-GFP (BioLegend, Cat# FM264G). For TEC staining, the thymus was cut into pieces, then was digested with Collagenase-V/DNase-I, as per previously published methods [51 (link)], then stained with surface molecules: fluorochrome-conjugated anti-CD45 (clone 30-F11), -MHC-II (clone M5/114.15.2), -Ly51 (clone 6C3), and -EpCAM (clone G8.8), which were purchased from BioLegend, and FITC-anti-Ovalbumin from AbCam (Cat# ab85584). Flow cytometry was performed using an LSRII flow cytometer (BD Biosciences) with 6 colors, and data were analyzed using Flow-Jo software.

Sections 5 μm in thickness were de-paraffinised and re-hydrated. A pretreatment method using heat-induced epitope retrieval was used, and the nonspecific binding was blocked with a protein blocker (DakoCytomation, Trappe, France) for 30 min at room temperature (RT). The sections were then incubated with the following primary antibodies against Ki67 (ab15580; Abcam), EpCam (clone G8.8; Biolegend), α-SMA (ab21027; Abcam), CD34 (RAM34, eBioscience), GP38 (MA615113; Thermo Fisher), CD24 (ab64064; Abcam), Lysozyme (ab108508; Abcam), Muc2 (Thermo Fisher) and ZO-1 (Invitrogen) antibodies.
The co-labelling with antibodies from the same species was performed using Opal™ Multiplex IHC kits (Akoya). After incubation with the primary antibodies, the slides were washed in PBS-T three times and probed with appropriated fluorescence-conjugated secondary antibodies for 1 h at room temperature. After three washings in PBS, the cell nuclei were counterstained by Vectashield mounting medium with DAPI (Vector).