I21414

Mouse hearts were fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, stained, and imaged, following standard immunohistochemistry and fluorescent microscopy methods, as previously described [40 (link),41 (link)]. An extra antigen retrieval step was added in all experiments by heating samples in a Tris-based buffer (pH 9) to 95 °C for 20 min. Primary antibodies against cardiac troponin T (cTnT) (AB8295, Abcam), Ki67 (AB16667, Abcam), GFP (SC-9996, Santa Cruz), FGF23 (SC-16849, Santa Cruz), Klotho (SC-22220, Santa Cruz), and Isolectin B4-biotin (I21414, Invitrogen) were used. Alexa Fluor conjugated secondary antibodies were purchased from ThermoFisher, Waltham, MA, USA). TUNEL staining was performed using an Apoptosis/Necrosis detection kit (Roche, Basel, Switzerland). Before mounting, the slides were incubated with 1% Sudan black (Sigma-Aldrich) in 75% ethanol at room temperature for 20 min to reduce tissue auto-fluorescence. Confocal images were acquired with an Olympus FLUOVIEW FV10i confocal microscope. Whole heart tissue section scanning was performed using TissueFAXS (TissueGnostics, Vienna, Austria). High magnification images of 10 fields of the cardiac tissue were randomly acquired with a light microscope (Olympus BX53). The number of Ki67+ cells or TUNEL+ cells in each image was determined with the Image J software (NIH, Bethesda, MD, USA).

The mice were perfused with PBS (G4202, Servicebio, Wuhan, China) and 4% paraformaldehyde (30525‐89‐4, Nanjing Chemical Reagent, Nanjing, China). After dehydration, permeation and embeddedness, the brain tissues were sliced by 10 μM thickness and placed in the same position of the six glass slides, and three brain slides are pasted on each glass slide. Next, dewaxing and hydration were carried out. The slices were blocked in 10% goat serum / PBST at room temperature and incubated with primary antibodies overnight at 4℃. The dilutions of primary antibodies were used in the experiments included as follows: NeuN (1:500, ab177487, Abcam, Cambridge, MA, USA), Iba1 (1:500, NB100‐1028, Novus, Centennial, Colorado, USA), CD45 (1:200, ab40763, Abcam, Cambridge, MA, USA), Ly6G (1:200, #31469, CST, Boston, USA), IB4 (1:200, I21414, Invitrogen, Carlsbad, CA, USA), NF‐κB (p65) (1:200, ab32536, Abcam, Cambridge, MA, USA), Nrf‐2 (1:200, 16396–1‐AP, Proteintech, Chicago, USA) and Fyn (1:300, ab125016, Abcam, Cambridge, MA, USA). The slices were washed by PBST and incubated with secondary antibodies (1:1000 dilution, Invitrogen, Carlsbad, CA, USA) for 1h at room temperature. DAPI (C3619‐PI19B, Southern Biotech, Birmingham, AL, USA) was incubated for 10 minutes. The quantification of images was analysed with Image J software as previously described.