Cas block

For immunofluorescence staining, embryos were fixed in 4% PFA for 1 h at RT on a rocking platform, followed by 2 washes in 1× PBS⁻ for 15 min each. For staining of LRO explants, embryos were dissected using a scalpel into anterior and posterior halves. Posterior halves (LRO explants) were collected and transferred to a 24-well plate and washed twice for 15 min in PBST. LRO explants and whole embryos were blocked for 2 h at RT in CAS-Block diluted 1:10 in PBST. The blocking reagent was replaced by an antibody solution (anti-acetylated tubulin antibody, diluted 1:700 in CAS-Block; c2181 Sigma) and incubated overnight at 4 °C. In the morning, the antibody solution was removed and explants/embryos were washed twice for 15 min in PBS⁻. The secondary antibody (diluted 1:1000 in CAS-Block; c2181 Sigma) was added together with Phalloidin (1:200) and incubated for a minimum of 3 h at RT. Before photo documentation, embryos or explants were briefly washed in PBS⁻ and transferred onto a microscope slide.

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.