Cd68 antibody

SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in 30% sucrose until cryosections (7 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany) and subjected to routine haematoxylin and eosin staining [24 (link)].
For CD68 IHC, SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in PBS at 4 °C until paraffin embedding. Sections (7 μm) were deparaffinized, and staining was performed using a Leica Bond MAX immunostainer (Leica Microsystems, Wetzlar, Germany) and CD68 antibody (1:1,000; Cell Signaling Technologies, Danvers, MA). To visualize neutral lipids and nuclei, cryosections (7 μm) were stained with ORO (Sigma-Aldrich, St. Louis, MO) and DAPI (Akoya Bioscience, Marlborough, MA), respectively. Images were acquired at 60 × magnification (Apo 60× Oil λS objective) using a Nikon Eclipse Ti microscope (NIKON Corporation, Tokyo, Japan) equipped with a Nikon A1 camera.

Heart slices from the four categories were also subject to IHC analysis. Briefly, the slides were incubated in 3% H₂O₂ solution for 10 min and antigen retrieval was performed by steam heating in 10 mM citrate buffer (pH 6.0) for 30 min. After epitope recovery, the slides were then treated with 10% of normal goat serum for 60 min, followed by incubation with active caspase 3 antibody (1:500, Cell Signaling Technology, Catlog #9664, MA, USA), CD31 antibody (1:1000, Cell Signaling Technology, Catlog #3528), and CD68 antibody (1:500, Cell Signaling Technology, Catlog #76437) incubation overnight at 4 °C. The slides were washed and incubated with secondary horseradish peroxide (HRP)-linked secondary antibody (Vector Laboratories, MN, USA) at 1:500 dilutions for 1 h. The samples were treated with the chromogen DAB for antigen detection and the final counterstaining was performed with hematoxylin.