Protease plus

In situ RNA hybridization was performed using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) according to manufacturer’s instructions. The following target probes were used: Mm-Dpt (Cat. #561511-C3), Mm-Tnfsf13b (Cat. #414891), Mm-Notch3 (Cat. #425171), Mm-Cxcl13 (Cat. #406311-C2). In brief, spleens were fixed in 10% formalin for 24 h at RT and embedded in paraffin. 3 μm-thick sections were baked in an oven at 60 °C for 1 h, then deparaffinized and dehydrated. Following rehydration, endogenous peroxidase activity was quenched with hydrogen peroxide for 10 min at RT. Target retrieval was carried out in a steamer (Braun, Type 3216) for 15 min at >98 °C. Protease treatment was performed with Protease Plus (Advanced Cell Diagnostics) for 20 min at 40 °C. Target probes were allowed to hybridize for 2 h at 40 °C, followed by signal amplification according to the ACD protocol. After the final amplification step, signal was detected with Opal520, Opal570 or Opal650-conjugated tyramide (Perkin Elmer). Sections incubated with a negative control probe (DapB) were analysed in parallel and a mix of positive control probes (Polr2a, Ppib, Ubc) was utilized to confirm RNA integrity. Images were acquired with an Olympus VS120 slide scanner fluorescence microscope using the VS-ASW-FL software (Olympus).

Samples from the left cranial and left caudal lung lobe together with spleen, kidney, liver, tracheobronchial and axillary lymph nodes, jejunum, colon, trachea, larynx and nasal cavity, were fixed by immersion in 10% neutral-buffered formalin and processed routinely into paraffin wax. Nasal cavity samples were decalcified using an EDTA-based solution prior to embedding. 4 µm sections were cut and stained with haematoxylin and eosin (H&E) and examined microscopically. In addition, samples were stained using the RNAscope technique to identify the SARS-CoV-2 virus RNA. Briefly, tissues were pre-treated with hydrogen peroxide for 10 min (room temperature), target retrieval for 15 min (98–101 °C) and protease plus for 30 min (40 °C) (Advanced Cell Diagnostics). A V-nCoV2019-S probe (Cat No. 848561, Advanced Cell Diagnostics) was incubated on the tissues for 2 h at 40 °C. Amplification of the signal was carried out following the RNAscope protocol using the RNAscope 2.5 HD Detection kit – Red (Advanced Cell Diagnostics).

RNAscope ISH assays were performed on formalin‐fixed CTC slides. The RNAscope probes Homo sapiens‐Hs‐panCK‐C3 (404751‐C3) and Homo sapiens 2.5 LS Probe‐Hs‐MKI67‐C3 (591778‐C3) were synthesized by Advanced Cell Diagnostics. The location and expression level of the target RNA were detected by the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, 320293) according to the manufacturer's instructions. In brief, the CTC slides were dehydrated in an ethanol gradient, treated with H₂O₂ for 10 min, and then treated with Protease Plus (Advanced Cell Diagnostics, 322000) at 40°C for 30 min. Next, the C3 probes were diluted in C1 probes at a 1:50 ratio for hybridization at 40°C for 2 h. Then, AMP1, AMP2, and AMP3 were added to the slides and incubated at 40°C for 30 min. After HRP treatment, C1 probes detected with TSA‐Plus FITC and C3 probes detected with TSA‐Plus Cy5 were added to the slides sequentially, with HRP blockers applied after every step. Finally, DAPI (Sigma‐Aldrich) was added to label the nuclei, and the slices were mounted. Proliferative CTCs were defined as DAPI+/Ki67+/CK+ cells, and regular CTCs were defined as DAPI+/Ki67–/CK+ cells.

The expression of Lgr5, Axin2, Rspo1, Rspo2, and Rspo3 was analysed by RNAscope (Advanced Cell Diagnostic, United States) in mice at E16.5 or E18.5. We analysed at least five mice embryos for each stage. Mice embryos were euthanised by decapitation and fixed in 4–10% PFA for a maximum of 48 h. After fixation, samples were decalcified in 10% EDTA for 3 days at 4°C, dehydrated by standard ethanol series followed by xylene. The samples were embedded in paraffin and sectioned at 5 µm. Tissues were processed using the RNAscope Multiplex Fluorescent v2 assay (cat. No. 323 110, Advanced Cell Diagnostics, United States). Samples were incubated in hydrogen peroxide (cat. No. 322 335, Advanced Cell Diagnostics, United States) at RT for 10 min before they were boiled in the target retrieval (cat. No. 322 001, Advanced Cell Diagnostics, United States) and pretreated with Protease Plus (cat. No. 322 331, Advanced Cell Diagnostic, United States). An LGR5 probe (RNAscope^® Probe Mm-LGR5, cat. No. 312171, Advanced Cell Diagnostics, United States) was used to detect transcripts, and DAPI (cat. No. 323 108, Advanced Cell Diagnostics, United States) was used for nucleus staining. Pictures were taken under a Leica DM LB2 microscope (Leica Microsystems, Germany) and processed by Adobe Photoshop 7.0.