Axiovert z1 microscope

Organoids were processed in the same way as for organoid formation efficiency assay. Cells were then plated using limiting dilution assay technique to have approximately 1 cell per 2.5 μL Matrigel drop. The drops were plated onto gridded 35 mm ibidi glass bottom dishes (Thermo Scientific, 81148) and overlayed with 1.2 mL of ExM. ExM was supplemented with 10 μM Y-27632 (Merck, 688000) for first 3 d of culture. Drops were screened and the relative positions of the drops containing one cell were stored using the Axiovision image software V4.8 and cells were imaged in phase contrast every 2 d using the Zeiss Axiovert Z1 microscope and Axio Observer software.

ISH for LRIG1 and AXIN2 were performed on 4 μm thick paraffin sections using RNAscope 2.0 High definition assay (Advanced Cell Diagnostics) following the manufacturer’s instructions. Briefly, the tissue sections were baked at 60 °C for 1 h and dewaxed with xylene, cleared in 100% ethanol and airdried. For tissue sections, the slides were treated according to the standard protocol: 10 min in Pretreat buffer 1, 15 min in Pretreat buffer 2 and 30 min at 37°C in Pretreat buffer 3. For organoid sections, milder treatments were necessary to avoid non-specific signal: Pretreat 2 for 5 min and Pretreat 3 for 15 min. Sections were then incubated with LRIG1 probe (Cat. no. 407421) or AXIN2 (Cat. no. 400241), positive control probe PPIB (Cat. no. 313901), negative control probe dapB (Cat no. 310043) for 2 h at 40°C. Positive and negative controls were performed for each sample. For the visualization of signal, the samples were incubated using the amplification kit and then treated with DAB for 10 min. Sections were then dehydrated, mounted in DPX (Sigma, 44581) and imaged using Zeiss Axiovert Z1 microscope and Axiovision imaging software SE64 V4.8.