Ab15580

Histology fresh frozen sections were fixed in 4% PFA and stained followed by hematoxylin and eosin staining. For immunofluorescence, pancreatic tumours were fresh frozen in OCT, fixed in 4% PFA or acetone, and stained according to manufacturer's protocol. Sections were stained using the polyclonal Ki67 (Rabbit Anti-Ki67, Abcam ab15580), Foxp3 (1054C, R&D ab15580), Trypsin 3 / PRSS3 (Goat anti-Trypsin, R&D Systems MAB8214), CD4 (GK1.5, in-house hybridoma supernatant), CD8 (5H10-1, BioLegend 100802), F4/80 (in-house hybridoma supernatant) and MECA-32 (Rat anti-PLVAP, in-house hybridoma supernatant). For immunofluorescence the following detection antibody were used: Donkey anti-Rabbit 488 (Molecular Probes), Donkey anti-Goat Alexa Fluor 546 (Life Technologies), Donkey anti-Rat 488 (Life Technologies) and DAPI (Life Technologies). Images were acquired using a Ziess LSM 780 confocal microscope.

Cells and tissue samples were fixed in 4% paraformaldehyde for 12 hours. The skin tissue samples underwent dehydration with 30% saccharose and were embedded into the optimal temperature compound (OCT) (Leica, 14020108926) and then cut into 10μm thick sections. The cells and sections underwent permeabilization with 0.05% Triton X‐100 (Sigma‐Aldrich, T8787) for 10 minutes at room temperature, blocking with 5% bovine serum albumin (BSA) (MP, 0218072801) at 37°C for 30 minutes, and incubated with the primary antibodies overnight at 4°C. Then, the cells and tissue sections were incubated with the related fluorescence secondary antibodies at room temperature for 1 hour. Finally, the nuclei were counterstained by Hoechst 33342 (Sigma‐Aldrich, 14533) for 10 minutes at room temperature. The images were obtained by a confocal microscope (Olympus, FV1000) and quantified by ImageJ software (NIH, USA). The antibodies involved in this study include the following: Ki67 (1:200, Abcam, ab15580), CD31 (1:100, R&D Systems, AF3628SP), KRT14 (1:400, Abcam, ab7800), α‐SMA (1:200, Abcam, ab5694) and Cy3‐conjugated IgG (Jackson, 111‐165‐003, 705‐165‐003 and 715‐005‐150).

For tissue preparation, placentas and embryos were fixed in 4% paraformaldehyde and processed either for routine paraffin embedding (placentas) or embedded in OCT for cryosectioning (embryos). Placental sections (7 µm) were deparaffinized and rehydrated according to standard protocols. Antigen retrieval was performed on placental sections with 2100 Antigen Retriever. Brain cryosections (10 µm) were warmed for 30 min. The cryo- and paraffin sections were blocked in PBS, 0.1% Tween 20 and 0.5% Bovine Serum Albumin, and incubated with tissue-specific antibody. Placental sections were incubated with antibodies against MCT1 (1:200; Sigma-Aldrich, AB1286-l, St. Louis, MO, USA) and MCT4 (1:100; Sigma-Aldrich AB3314P). Brain sections were incubated with antibodies against Ki67 (1:3000; Abcam, ab15580) ), SOX2 (1:200; R&D Systems AF2018, Minneapolis, MN, USA), PAX6 (1:200; Biolegend 901301, San Diego, CA, USA) and NESTIN (1:200; Abcam ab6142, Cambridge, UK). Detection was carried out with the appropriate AlexaFluor488 or 568-conjugated secondary antibody, respectively (1:300; Thermofisher, Waltham, MA, USA). Nuclear counterstaining was performed with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (MilliporeSigma, 10236276001, Burlington, MA, USA).