Hcx pl apo 40 1.25 objective

Immunofluorescence experiments were performed on 4% PFA-fixed cells grown on fibronectin-coated coverslips using standard procedures. Images were acquired using a Leica TCS SP5 confocal microscope with HCX PL APO × 40/1.25 objective. The presented images are the maximal projections of the five central stacks of the total z-axis range acquired (between 12 and 18 μm, each stack was 0.5 μm size).

Whole spheroids were harvested and fixed in 4% PFA with 8% sucrose overnight, then washed 3× in PBS for 1 h. Spheroids were processed for immunofluorescence according to the following conditions: permeabilization and blocking with B-PBT (1% Triton X-100, 10% FBS and 4% BSA in PBS) for 2 h at RT, followed by incubation with primary antibody (Supplementary Table 6) in B-PBT overnight a 4 °C. Then two washes for 2 h in 0.2% PBT (0.2% Triton X-100 in PBS) and one wash in B-PBT for 2 h before incubating in secondary antibody in B-PBT overnight at 4 °C. After two washes in 0.2% PBT and one wash in PBS, cell nuclei were counterstained with DAPI. Images were acquired using a Leica TCS SP8 confocal microscope with HCX PL APO ×40/1.25 objective.

ESCs were fixed in 4% paraformaldehyde (PFA) for 20 min, washed, blocked and incubated with primary antibodies from different species following the protocol procedure. PLA assay (Olink Bioscience) was performed by incubating secondary antibodies conjugated with oligonucleotides (probes) for 1 h at 37 °C followed by hybridization and ligation with the PLA probes. Amplification of the occurred ligation was performed by in situ amplification in the presence of fluorescent-labelled oligonucleotides. Images were acquired using Leica TCS SP5 confocal microscope, with HCX PL APO × 40/1.25 objective. The signal intensity of the proximity reactions was quantified using Volocity Analysis software (PerkinElmer).