Cas block solution

The liver tissues were fixed overnight in 4% PFA, embedded in paraffin or O.C.T compound (Leica) and sectioned sequentially at 8–15 μm. The sections were permeabilized with 0.2% Triton X–100 and blocked with CAS–Block solution (Invitrogen) to prevent non–specific binding. For immunofluorescence staining analysis, the sections were incubated with primary antibodies diluted in CAS–Block solution overnight at 4°C. After the primary antibody incubation, the sections were washed with DPBST three times for 10 min each. The following secondary antibodies were diluted in PBS and applied for 60 min. Images were visualized by fluorescence microscopy (Leica). The primary antibodies used for analysis are listed in S3 Table. The nucleus was stained with hematoxylin (Sigma) and blued in 0.2% ammonium hydroxide. Tissue was then placed in Eosin Y solution (Sigma) and followed by several washings and dehydrating. Slides were mounted with a mounting solution (Leica).

Differentiated EBs (D8) were cultured on gelatinized 12-mm glass cover slips, fixed for 30 min at room temperature in 4% paraformaldehyde, and permeabilized for 30 min at room temperature using 0.2% Triton X-100. After permeabilization, the EBs were blocked for 10 min at room temperature with CAS-Block solution (Invitrogen), and incubated overnight at 4 °C with mouse monoclonal antibodies against sarcomeric α-actinin (Abcam), cardiac troponin T (cTnT, Abcam), or connexin 43 (Cx43, Abcam). Additionally, goat anti-mouse IgG (Abcam) was used as the primary antibody to label EBs. Confocal images were obtained using a Carl Zeiss LSM 700 laser-scanning microscope (Zeiss, Oberkochen, Germany) [19 (link)].

The immunofluorescent labeling of iHLOs or iHEAs was performed. Briefly, samples were fixed with 4% paraformaldehyde overnight after DPBS washing. Fixed iHLOs or iHEAs were incubated with 15% sucrose solution until they sunk, and then they were left in a 30% sucrose solution overnight. iHLOs or iHEAs were removed from the 30% sucrose solution, rinsed with an OCT compound, and snap-frozen embedded in OCT. Cryomolds were stored at −80 °C until cryosectioning. Cryomolds were sequentially sectioned at 10 μm using a cryostat (CM1950, Leica, Buffalo Grove, IL, USA). Sectioned slides were stored at −80 °C until use. To immunolabel the iHLO or iHEA slides, the slides were permeabilized with 0.1% Triton-X 100 for 30 min and blocked with a CAS-Block solution (Invitrogen, Carlsbad, CA, USA) for 30 min. After blocking, the samples were incubated with the primary antibodies listed in Table S2 overnight. The secondary antibodies were diluted in PBS and applied for 1 h: Alexa Fluor 488/594 anti-mouse IgG, IgG1, anti-goat IgG, anti-rabbit IgG (Invitrogen, 1:1000), and FITC anti-hamster IgG (Jackson ImmunoResearch Lab, 1:1000). Nuclei were stained with DAPI (Invitrogen, Carlsbad, CA, USA).

To generate brain metastasis, mice were inoculated intracardially as previously described [6 (link)]. Six weeks following the inoculation, mice were perfused with 4% paraformaldehyde, sacrificed, and brains were fixed and embedded in optimal cutting temperature compound (Tissue-Tek^® O.C.T., Sakura Finetek USA, Inc., Torrance, CA, USA).
OCT-embedded 10 µm brain sections were cut. The sections were blocked for 30 min. with CAS-Block^TM solution (008120, Life Technologies, Carlsbad, CA, USA). Primary antibodies (Abs) were incubated in Ab diluent (ab64211, Abcam, Cambridge, UK) with 0.02% Triton X-100 overnight at 4 °C, followed by fluorescently conjugated secondary Abs in 0.02% Triton X-100 in PBS for 60 min at RT. Coverslips were mounted using 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount (Southern Biotech, Birmingham, AL, USA). The images were acquired with a × 40/1.10 water objective, using a Leica SP8 confocal microscope and Leica SP8 software (LAS-X v3.5.7.23225, Leica Microsystems, Wetzlar, Germany).
Information on primary Abs against JunB, Iba-1, and melanoma markers is detailed in Table S1.

Mouse hind paws were isolated after neurobehavioral tests. Dissected tissues were fixed with 10% paraformaldehyde overnight and cryoprotected for 72 h with 30% sucrose in PBS at 4 °C. The samples were embedded in OCT (#CM 3050S; Leica) and sliced into 30 μm thick sections. The slides were washed with 0.05% Triton X-100 in PBS for 4 h and incubated with CAS-Block^TM solution (#008120; Life Technologies) at room temperature for 1 h. The primary antibodies used were rabbit anti-PGP9.5 (1:100; #ab108986, Abcam) and goat anti-Collagens VI (1:100, #AB769; Chemicon). For double staining, two antibodies from different species were mixed and incubated overnight with the sample sections at 4 °C. After washing away the primary antibodies with PBS, the tissues were incubated with secondary antibodies (Alexa-488, 594, or 647, 1:200; Invitrogen) and Hoechst 33342 (10 mg/mL, #H1399; Invitrogen) for 2 h at room temperature. Immunofluorescence images were acquired using a multiphoton laser scanning microscope with a 40× objective (FV1000MPE, Olympus).