Anti gr 1

The following primary antibodies were from Cell Signaling: anti-phospho-Smad3; anti-Smad3; anti-phospho-ERK MAPK; anti-ERK MAPK, and anti-M-CSF Receptor. The FC blocker used was Mouse SeroBlock FcR, which is a rat monoclonal antibody (clone FCR 4G8, BioRad) that specifically recognizes mouse CD16 and CD32, which are cell surface proteins also known as FcRgIII and FcRgII, respectively. Mouse monoclonal anti-K2, clone 3A3 (1:2000, EMD Millipore); anti-F4/80 (1:50, eBioscience); anti-Gr-1 (1:50, AbD Serotec); anti-CD206 (1:50, BioRad); goat horseradish peroxidase-conjugated anti-mouse IgG (1:2,000) and goat horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000) were from Calbiochem. Vecta-shield with 4′,6-diamidino-2-phenylindole was from Vector Laboratories. Gel electrophoresis reagents were from BioRad. CSF-1 was from Thermo Scientific. LPS and CCL2 were from Sigma.

Tissues were collected at the designated times and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek), and 8-μm sections were prepared. Macrophages were detected with rat anti-F4/80 (eBioscience). M2 Macrophages were detected with rat anti-F4/80 and rat anti-mouse CD206 (BioRad). Monocytes/neutrophils were detected with anti-Gr1 (AbD Serotec), followed by Alexa 488 or Alexa 568-conjugated goat anti-rabbit IgG. Stained sections were analyzed using fluorescent or bright-field imaging microscopy (Leica) and ImagePro Plus Capture and Analysis software (Media Cybernetics). F4/80-, CD206-, and Gr1-positive areas were quantified in 15 independent fields/section using Image Pro-Plus software (25 (link), 27 (link)).