Ds qilmc

Paraffin-embedded tumor issues were sliced into 5-mm thick section and tissue slides were deparaffinized in Tissue-Clear (1466; Sakura) and rehydrated with sequential immerse in 99–95–70% ethanol. Frozen tissue samples embedded in the OCT Embedding Compound (SAKURA) were sectioned using a cryostat, followed by fixation with 4% PFA for 20 min. Tissue slides were stained with a rat anti-mouse endomucin (1:400; 14-5851-85; eBioscience) antibody, a mouse anti-human αSMA (1:200; M0851; clone 1A4; DAKO) antibody, a rabbit anti-mouse NG2 (1:400; AB5320; Millipore) antibody, and a rat anti-mouse Ki67 (1:100; M7248; DAKO) antibody, followed by staining with species-matched secondary antibodies as follows: an Alexa Fluor 555-labeled goat anti-rat (1:400; A21434; Invitrogen), an Alexa Fluor 488-labeled donkey anti-mouse (1:400; A21202; Invitrogen), and an Alexa Fluor 488-labeled donkey anti-rabbit (1:400; A21206, Invitrogen). Positive signals were detected using a fluorescence microscope equipped with a camera (Nikon, DS-QilMC). Images were analyzed using an Adobe Photoshop software (CS5; Adobe) program. Vascular coverage was quantified as a percentage of vessels covered by αSMA⁺ cells by calculating overlapping area of endomucin and αSMA⁺ positive signals.

All zebrafish work was performed at the Zebrafish Core Facility of the Karolinska Institutet and experiments were conducted under the regulation of the facility. Wild‐type AB strains of zebrafish were raised at 28 °C under standard conditions. At 24 h‐post‐fertilization (hpf), zebrafish embryos were transferred to an E3 medium containing 0.2 mmol L⁻¹ 1‐phenyl‐2‐thiourea (Cat. 189 235, Sigma‐Aldrich) to prevent pigmentation. Embryos were anesthetized with 0.04 mg mL⁻¹ of tricaine (Cat. MS‐222, Sigma‐Aldrich) at 48 hpf prior to microinjection. CAFs isolated from Bmal1^−/− and Bmal1^+/+ mice were labeled with 1 µg mL⁻¹ 1′‐dioctadecyl‐3, 3, 3′, 3′‐tetramethylindocarbocyanine per‐chlorate (DiI, Cat. 42 364, Merck) in vitro. Approximately 100–200 CAFs and 100–200 GFP tumor cells at the ratio of 1:1 were subsequently injected into the perivitelline space of each eligible embryo. Zebrafish embryos were transferred into 24‐well plates (1 embryo per well) and incubated at 33 °C for additional 48 h. Micrographic images were taken at 48 h after cell injection. Positive signals were detected by using a fluorescence microscope equipped with a camera (Nikon, DS‐QilMC). Images were analyzed by using an Adobe Photoshop software (CS6, Adobe) program.

Paraffin-embedded tissues were cut into 5-μm slides. Slides were baked for 1 h at 60 °C and deparaffinized in Tissue-Clear (1466, Sakura), and sequentially rehydrated in 99%, 95% and 70% ethanol and counterstained with H&E. Mounting was performed with Pertex (0081, HistoLab). Deparaffinized slides were boiled for 20 min in an unmasking solution (H3300, Vector Labs) then subsequently blocked with 4% serum. Tissues were incubated with a mouse anti-human mtTFA antibody (1:200 dilution; 119684, Abcam) and a mouse anti-human MTCO2 antibody (1:200 dilution; 110258, Abcam) at 4 °C overnight, followed by staining with a species-matched secondary Alexa Fluor 555-labelled donkey anti-mouse (1:400 dilution; A-31570, Thermo Fisher Scientific) and a DAPI (10236276001, Roche). Slides were mounted with Vectashield (H-1000, Vector Labs). Signals were detected by fluorescence microscope equipped with a camera (Nikon, DS-QilMC). Images were analysed using an Adobe Photoshop software (CC 2019, Adobe) programme.