Immunofluorescence Analysis of FSTL1, IL-1β, and TNF-α

Formalin-fixed and paraffin-embedded (FFPE) sections were deparaffinized and rehydrated before antigen retrieval and blocking. Sections were co-incubated with the antibodies for FSTL1 (1:200, Abcam, Cambridge, MA, UK) and IL-1β (1:200, Abcam) or FSTL1 and TNF-α (1:200, Abcam). As a secondary antibody-only control, PBS was used instead of primary antibodies. After overnight incubation at 4°C, slides were washed with PBS and incubated with secondary antibodies (1:400, Alexa Fluor 488 donkey anti-goat IgG, Molecular Probes, or Rhodamine 568-labeled anti-rabbit IgG, Chemicon International, Temecula, CA, USA) for 1 h at room temperature. Finally, nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI) and sections were examined under a confocal laser scanning microscope (Fluoview FV1000-D, Olympus, Tokyo, Japan). H & E staining for histology analysis was performed as described [35 (link)]. Immunohistochemistry staining was done as we described before [36 (link)].