Sc 20150

The following antibodies were used for immunostaining. Anti-Arginase-1 (Santa cruz, sc-20150, 1:200), anti-BMP-2 (Abcam, ab6285, 1:100 or Bioss, bs-1012R, 1:100), anti-β-catenin (Abcam, ab2365, 1:100), anti-caspase-3 (Epitomics, 1476−1, 1:100), anti-F4/80 (Abcam, ab6640, 1:100), anti- HSP47 (Novus, NBP1-97491, 1:50), anti-K15 (Abcam, ab52816 or ThermoFisher, MA5-11344, 1:100), anti-Ki67 (ThermoFisher, RM-9106, 1:100), anti-perilipin-1 (Abcam, ab3526, 1:100), and anti-phospho-Smad1/5 (Cell signaling, 9516, 1:50).
The following antibodies were used for flow cytometry or cell sorting. Anti-CD45 (BD pharmingen, 553081, 1:200), anti-CD86 (eBioscience, 17-0862, 1:400), anti-CD206 (BioLegend, 141706, 1:40), and anti-F4/80 (eBioscience, 17-4801, 1:10). The corresponding isotype controls were APC/Cy7 Mouse IgG2a, κ (BioLegend, 400230), FITC Rat IgG2a, κ (BD pharmingen, 557228), PE Rat IgG2a, κ (BD pharmingen, 557229), and APC Rat IgG2a, κ (BD pharmingen, 551442).

Total protein was harvested and resuspended in 1% SDS from cultures of 1 × 10⁶ BMDMs and IC-21 using TRIzol Reagent (Invitrogen) per manufacturers protocol and quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 10–20 μg of protein was subjected to SDS-PAGE, transferred to a PVDF membrane, blocked in 5% Protein Blocking Reagent (Azure) for 1 hr at RT and then probed overnight with primary antibodies at 4 °C. Antibodies included M(LPS/IFNγ) markers iNOS (abcam ab3523; 1:1,000) and MCP1(CST 2029S; 1:500), M(IL-4/IL-10) markers ARG1 (Santa Cruz sc-20150; 1:200), CD206 (abcam ab64693; 1:500), macrophage marker CD68 (abcam ab53444; 1:500) and loading control Gapdh (abcam ab8245; 1:1,000). The following day, blots were washed three times in Protein Wash Buffer (Azure) and probed with HRP-conjugated secondary antibody of the appropriate species at 1:2,500 for one hour at room temperature. Blots were then washed three times in Protein Wash Buffer and once with TBS, developed with Radiance Plus ECL Reagent (Azure), and imaged on the c600 Imager (Azure).