Anti cd68

Infarcted hearts were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 5 μm intervals from the level of coronary artery ligation to the heart apex. After routine hydration, the heart section was microwaved for 3 min in citrate buffer (0.4 g/L citric acid and 3 g/L sodium citrate) for antigen retrieval. Permeabilization and blocking were performed in PBS with 5% bovine serum albumin (BSA) and 0.2% Triton X-100, at room temperature for 30 min. The sections were incubated overnight at 4 °C with the following primary antibodies: anti-HIMF (Cat#ab39626, Abcam, Cambridge, MA, USA), anti-CD68 (Cat#NBP-33337, Novus, CO, USA), anti-NOS2 (Cat#2D2-B2, R&D system, Minneapolis, MN), and anti-Arg1 (Cat#93668, Cell Signaling Technology, Danvers, MA, USA. Then the samples were incubated for 1 h at 30 °C with the following respective secondary antibodies: A-21070 (1:400), A-11006 (1:400), A11030 (1:400) (Invitrogen, Carlsbad, CA) and 4′,6–Diamidine–2′–phenylindole dihydrochloride (DAPI, Cat#10236276001, Sigma-Aldrich, Burlington, MA, USA). Confocal images were captured under a Zeiss LSM880 microscope (Carl Zeiss, Germany). A total of 3–4 animals were examined per group.

For histology, tissues were fixed in 10% neutral buffered formalin or Carnoy’s fixative (60% methanol, 30% chloroform, and 10% acetic acid) before paraffin embedding. Images were obtained with an Eclipse i80 microscope (Nikon Instruments, Melville, NY, USA). Paraffin-embedded 5-μm-thick sections were stained with H&E for gross morphology and with Alcian blue to detect goblet cells. Immunohistochemistry and immunocytochemistry were performed with appropriate antibodies on paraffin-embedded sections [18 (link), 21 (link)]. Antibodies used were rabbit anti-DCLK1 (1:200, ab31704) and Anti-CR (1:250, ab37056) from Abcam, Cambridge, UK; mouse anti-Notch1 NICD (1:200, clone OTI3E12) from Origene, Rockville, MD; rabbit anti-Hes1 (1:200, #11988) and mouse anti-Ki-67 (1:200, #9449) from Cell Signaling Technology, Danvers, MA; anti-CD68 (1:200, NB600-985) from Novus Biologicals (Littleton, CO, USA), Anti-Muc2 and anti-NE (1:200) Santa Cruz, Dallas, TX; Anti-CD11c and anti-F4/80 (Thermo Fisher), and anti-Ly6G (R&D, Minneapolis, MN). Antibody controls included omission of the primary antibody or detection of endogenous IgG staining with goat anti-mouse or anti-rabbit IgG (Calbiochem, San Diego, CA, USA).