Superfrost plus adhesion microscope slides

Samples were processed using a tissue processor (Leica, #ASP300) and paraffin-embedded samples were cut into 5 μm sections using a microtome (Leica, #RM2255) and mounted on Superfrost Plus Adhesion Microscope Slides (Epredia™, # 12302108). Sections were initially subjected to consecutives washes with xylene and ethanol for deparaffinization and finished by washes with deionised water. Endogenous peroxidase activity was blocked in 1% hydrogen peroxide (H₂O₂) for 30 min. Antigen retrieval was conducted by incubating in Pepsin Reagent solution (Sigma-Aldrich #R2283) at room temperature for 15 min. The samples were then blocked for 1 h with 10% serum of the secondary antibody species at room temperature. The samples were subsequently incubated with primary antibodies (Supplementary Table 2), in 10% serum, overnight at 4 °C, and then incubated 1 h with secondary antibodies diluted 1:200 (Supplementary Table 2). The secondary antibody signal was amplified by incubation with Vectastain Elite ABC Reagent (Vector labs, PK-6100) for 30 min. The signal was developed using SIGMAFAST™ 3,3′-Diaminobenzidine (DAB) (Merck, #D4418) for up to 10 min at room temperature. Samples were counterstained incubating for 30 s in a Mayer’s Modified Haematoxylin solution (Abcam, #220365).

Mice were euthanized with CO₂ and transcardially perfused with 20 mL Dulbecco's Balanced Salt Solution (DPBS, Thermo Scientific #14190144) followed by 20 mL 4% (wt/vol) paraformaldehyde in DPBS to fix the tissue. Brains were isolated, snap frozen in -40 to -60 °C cold isopentane and stored at − 80 °C. Brains were transferred to − 20 °C 2 h before 8 µm thick coronal sections were obtained. Slices from CTRL, cKO and BK^−/− were collected on the same glass slides (epredia Superfrost™ Plus Adhesion Microscope Slides #J1800AMNZ) together with additional sections from BK^+/+ that served as positive controls. Blocking buffer (BB) consisting of DPBS supplemented with 2% Glycerol, 5% NGS, 0.3% Triton-X-100, 2% (wt/vol) BSA and 50 mM NH₄Cl was used for permeabilization and blocking of unspecific antibody binding. Primary antibodies against BK were diluted 1:1000 in BB and incubated overnight at 4 °C. Sections were then washed thrice with 0.01% Triton-X-100 in DPBS and blocked again with BB for 1 h before incubation with secondary antibody (1:2500) and Hoechst 33342 (1:1000) for 2 h. After three washes with 0.01% Triton-X-100 in DPBS, DPBS and water, sections were mounted with Permafluor (Fisher Scientific, #TA-030-FM). Primary antibodies are listed in Table S7.

As the polymer structure of the embedded HistoGel differs substantially from that of the paraffin, organoid detection within the HistoGel was easily possible during sectioning. FFPE blocks were sectioned at a thickness of 3.5 µm and transferred on Superfrost Plus Adhesion microscope slides (J1800AMNZ, Epredia). Slides were incubated in a slide oven overnight at 37 °C, with the specimen facing up to prevent potential loss of organoids due to melting paraffin.

To obtain frozen sections, fixed brains were cryoprotected in 30% sucrose in PBS and embedded into Killik O.C.T. (Bio Optica, Milan, Italy) and then placed at −80 °C for quick freezing. Brains were sectioned into 12 µm-thick slices using the HM525 NX cryostat (Epredia, Portsmouth, NH, USA) and finally collected onto polarized SuperFrost™ Plus Adhesion Microscope Slides (Epredia).
ISH on frozen tissue sections was performed as described in Gabellini et al. [25 (link)], with some modifications. Cryosections were thawed and washed in PBT (PBS + 0.5% Triton X-100) and incubated with either 300 ng/mL prr21a-201 or prr12b-201 antisense probes at 65 °C O/N. Slides were then rinsed at 65 °C in Hybe Wash and MAB + 0.1% Tween20 (Sigma-Aldrich) (MABT) solutions at RT. After 1 h-long equilibration at RT in the previously described blocking solution added with 20% lamb serum, slides were incubated with anti-DIG antibody (diluted 1:2500 in the blocking solution) at 4 °C O/N. Slides were stained in BM Purple Solution in the dark at RT. After the staining procedure, images were acquired using a Nikon Eclipse Ti microscope (Nikon Instruments).

Preparation of tissue microarrays and immunohistochemical staining were performed as previously described [24 (link)]. The intensity of COMP was evaluated independently by two researchers in a blinded fashion using scores: 0 for negative staining, 1 for low expression, 2 for moderate expression and 3 for high expression. Staining in cancer cells was evaluated separately from the stroma.
To evaluate metastases into the lungs of mice, the first fifteen 5 μm cuts were discarded. The next tissue cut was mounted on Superfrost Plus Adhesion Microscope slides (Epredia). The antigen retrieval was performed with the heat-induced epitope retrieval method using 10 mM citrate buffer (pH = 6). Human metastatic cells were stained with an anti-pan-cytokeratin antibody (Sigma) followed by a Signalstain Boost IHC detection reagent (Cell Signaling Technology). ImmPACT DAB (Vector Laboratories) was then used for the detection of positive cells. The final mounted samples were scanned with the Aperio Scanner system (Leica) at 40X, and the number of positive cells was evaluated by QuPath software [25 (link)].