Ab5694

Tissue from mouse liver was collected, fixed with 4% paraformaldehyde, embedded and frozen in Tissue-Tek^® O.C.T.™ Compound and cut to yield 7 µm sections. Frozen liver sections were incubated with the following primary antibodies: desmin (1:200, R&D, AF3844), CD31 (1:100, Abcam, ab28364), F4/80 (1:100, Thermo, BM8), HNF4a (1:100, Cell Signaling, #3113S), cytokeratin (1:200, TROMA-III), aSMA (1:100, Abcam, ab5694), Thy1.2 (1:100, Thermo, 53-2.1) and Slit2 (1:100, Abcam, ab7665). Fluorescent secondary antibodies with different fluorescent conjugates (goat anti-rat 488 (Invitrogen, A11006), 1:200; goat anti-rabbit 488 (Invitrogen, A11034), 1:200; donkey anti-rabbit Cy5 (Novus Biologicals, NBP1-75286PECY55), 1:100; donkey anti-goat 488 (Invitrogen, A11055), 1:200) were employed followed by Hoechst 33258 staining (Sigma, B2883-100MG). All immunohistochemistry- and immunofluorescence-based quantification was performed on sections containing representative tissue from several lobes of the liver (three midsized tissue pieces per liver per mouse). Fluorescence images were captured employing a Nikon eclipse Ti2 microscope or DMi8 confocal laser microscope (Leica). Images were analyzed using ImageJ software (Version 1.51n).

All tissues or matrices, including patient TMAs, were fixed in 10% buffered formalin and embedded in paraffin. The 4-μm sections of samples were deparaffinized with xylene and rehydrated in graded ethanol washes. For immunohistochemistry, Bond Dewax Solution (AR9222) was used. Antigen retrieval was performed using Bond RX H2(30) protocol: HIER 30 min ER1 (citrate pH 6) OR ER2 (EDTA pH 9) at 100 °C. Slides were quenched in BOND Polymer Refine Detection (Leica Biosystems DS9800). Slides were incubated with primary antibodies (Supplementary Table 3) α-SMA (Abcam ab5694, 1:100 dilution), Ki67 (Thermo Fisher RM-9106-511, 1:500 dilution), pan-cytokeratin (Leica-Novostra C11, 1:50 dilution), PDGFR-β (Cell Signaling 3169 1:100 dilution), pMLC2 (Cell Signaling 3675S 1:100 dilution), CD31 (Taylor Bio-Medical DIA-310 1:100 dilution), pSTAT3 (Tyr705) (Cell Signaling 9131S 1:100 dilution), MPO (Agilent A039829-2 1:2,000 dilution), F4/80 (Abcam 100790 1:100 dilution), CD8 (Cell Signaling 98941,1:200 dilution) and CK19 (Abcam 133496) 1:1,000 dilution), followed by visualization by diaminobenzidine (DAB). H&E staining and counterstaining were performed on a Leica autostainer. Antibodies were validated by suppliers. Slides were digitized on an Aperio slide scanner and representative areas per tumor acquired and analyzed by researchers blinded to the conditions of the experiment.

Tissues were dissected at the appropriate gestational or postnatal stage. Tissues were fixed in 4% PFA at 4°C, dehydrated, and embedded in paraffin wax for sectioning. 4–6μm sections were used in all experiments. TBX18-2 antibody is used at 1:800 in normal goat serum blocking solution and incubated overnight at 4°C. Following washes, a Goat anti-Rabbit:HRP (Jackson ImmunoResearch 111-035-003 1:500) secondary antibody was applied for 1hr at room temperature. Sections were covered in TSA-Rhodamine for 6 minutes then washed and counter stained with Hoecsht 33342. Commercial antibodies are VIM (Abcam ab92547 1:500), CK8/CK18 (ab53280 1:100), CK5 (ab24647 1:750), and SMA (ab7817 1:75, ab5694 1:1000) with secondary antibodies (from Thermo-Fisher Scientific), Goat anti-Rabbit IgG, Alexa Fluor 488, A-11008 1:200 and Goat anti-Mouse IgG, Alexa Fluor 568, A-11004 1:200 and Alexa Fluor 647, A-31573, 1:200.