Anti cd68 antibody

Paw and joint knee sections were immunostained with CD68 as previously described by Cordaro and coll [39 (link)].. Slices were incubated overnight with anti-CD68 antibody (1:100 in PBS, v/v, Santa Cruz Biotechnology, Heidelberg, Germany). All immunohistochemical analyses were carried out by an observer blind to treatments.

Anti-phospho Smad2 (pSmad; Cat#AB3849), anti-Ki-67 (Cat# AB9260), HRP-conjugated anti-rabbit IgG (Cat# 12–348), and anti-vascular endothelial growth factor (VEGF; Cat# 05–443) were purchased from Millipore (Billerica, MA). Anti-caspase-3 antibody (Cat# 9661) was from Cell Signaling Technology (Danvers, MA). Anti-CD68 antibody (Cat# 6A324) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti- hypoxia-inducible factor 1-alpha (HIF-1α; Cat#NB-100-449) was purchased from Novus Biologicals (Littleton, CO). Anti-occludin (Cat#331500) and anti-ZO-1 (Cat#617300) antibodies were purchased from Invitrogen. Anti-granulocyte receptor-1 (Gr1; Cat# MBS520313) was from MyBioSource (San Diego, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cat# A4116), Cy3-conjugated anti-rabbit IgG (Cat# C2306) and anti-β-actin (Cat# A5441) antibodies were purchased from Sigma-Aldrich. Anti-myeloperoxidase (MPO) (Cat#DOM00001G), anti-claudin-3 (Cldn3; Cat# 341700), AlexaFlour 488-conjugated anti-mouse IgG (Cat# A11029), AlexaFluor 488-phalloidin (Cat# A12379), AlexaFluor 546-phalloidin (Cat# A22283), and anti-TNFα (Cat# AMC3012) antibodies were purchased from Thermo Fisher Scientific.

The dissected lungs were fixed in PBS/4% paraformaldehyde (PFA) (Sigma) for 48 hours, dehydrated with graded ethanol (30%, 50%, 75%, 95%, 100%) and embedded in paraffin. Sections (4 µm) were blocked with PBS/6% goat serum (Gibco) at room temperature for 45 minutes, incubated with anti‐CIRP antibody (Proteintech), anti‐CD31 antibody (Abcam), anti‐CD68 antibody (Santa Cruz), anti‐SMA antibody (Genetex), anti‐CAV1 antibody (Abcam) or anti‐CAVIN1 antibody (Abcam) at 4°C overnight and washed with PBS/0.1% Tween (PBST). Subsequently, for diaminobenzidine (DAB) staining, the sections were incubated with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (Beyotime) at room temperature for 30 minutes and then treated with DAB peroxidase substrate solution (Beyotime) according to the manufacture's instruction for colour development. For Alexa Fluor staining, the sections were incubated with Alexa Fluor 488 conjugated goat anti‐rabbit IgG (Abcam) or Alexa Fluor 555 conjugated goat anti‐mouse IgG (Abcam) at room temperature for 1 hour. Nuclei were stained with DAPI (Sigma). The images were captured with fluorescence microscope (Leica).