Cas block

Tumours harvested from mice were processed as formalin-fixed paraffin-embedded (FFPE) with 4 µm sections, dewaxed and antigen retrieval performed using Tris-EDTA pH9.0 prior to tissues being blocked with 5% normal goat serum (NGS) in CAS-Block (Life Technologies) for 30 min at room temperature. Primary antibody (anti-CD3 antibody) (1:700; Cell Marque (Sigma-Aldrich)) diluted in CAS-Block plus 5% NGS was added to the tissues and incubated overnight at 4 °C. After washing, secondary antibody Alexa-555 (goat anti-rabbit, 1:500, ThermoFisher Scientific) was prepared in CAS-Block with 5% NGS and incubated for 2 h at room temperature. Slides were counterstained with DAPI (1:2000, ThermoFisher Scientific) and imaged using the Zeiss Axio Scan.Z1 slide scanner.

Cells were grown on glass slides and fixed and permeabilized in cold (−20 °C) methanol. Cells were blocked overnight in CAS-block (Life Technologies, Carlsbad, CA), then incubated overnight with primary antibodies diluted 1:200 in CAS-block, then washed and incubated overnight in secondary antibodies diluted 1:500 in 5% BSA PBS. Cells were then washed, treated for 5 min with DAPI, and covered with coverslips. A confocal laser scanning microscope (Zeiss Axiovert 200M; Carl Zeiss MicroImaging, Thornwood, New York) was used to obtain images. Images were acquired and processed using the Olympus FluoView FV1000 software.

Five-micrometer-thick FFPE tissue sections were dewaxed and rehydrated through xylene and water-based ethanol solutions. Heat-induced epitope retrieval was performed with a pressure cooker (2100 Antigen Retriever, BioVendor, Brno, Czech Republic) in citrate or Tris-EDTA buffer (Agilent Dako, Santa Clara, CA, USA). Following endogenous peroxidase quenching (BLOXALL, Vector Laboratories, Burlingame, CA, USA), tissues were incubated with CAS-block (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature (RT) and Ultra V block (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. Primary antibodies (Table S3) diluted in CAS-block were applied for 30 min, followed by UltraVision ONE HRP polymer (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, at RT. The ImmPACT DAB substrate (Vector Laboratories, Burlingame, CA, USA) was applied. Tissues were counterstained with hematoxylin, dehydrated, and mounted with Cytoseal 60 (Thermo Fisher Scientific, Waltham, MA, USA). Imaging was performed with an automated BX63 microscope connected to a DP-80 camera (Olympus, Tokyo, Japan).

Co-injected fluorescein dextran (70,000 MW, ThermoFisher, D1822) was used as lineage tracer for dorsal lips in IF analyses. For IF analyses, embryos were fixed in 4% paraformaldehyde for 1 h at RT, followed by two washes in 1× PBS for 15 min each. For staining of animal caps or bisected specimens, embryos were manually dissected horizontally or sagittaly after fixation, transferred to 24-well plates, and washed twice for 15 min in PBST (PBS/0.1% Triton X-100). After blocking for 2 h at RT in CAS-Block (1:10 in PBST; ThermoFisher, #008120) blocking reagent was replaced by antibody solution (diluted in CAS-Block) for incubation ON at 4°C. Antibodies used were: β1-Integrin (DSHB 8C8-s; 1:70), MZ15 (DSHB; 1:200). Then antibody solution was removed and explants washed twice for 15 min in PBS. Secondary antibodies (ThermoFisher, all 1:1000 in CAS-Block) were incubated for 2 h at RT. Cell borders were visualized using AlexaFluor^TMPlus 405 Phalloidin (ThermoFisher A30104) overnight at 4°C (1:100 in PBS-). For photo documentation, bisected embryos or caps were transferred onto microscope slides or positioned in low melt agarose on a Petri dish (0.5% low melt agarose in 1× PBS-).