Rat anti f4 80 monoclonal antibodies

Immunofluorescence staining was performed to identify macrophages and their subtypes. Total macrophages were stained with rat anti-F4/80 monoclonal antibodies (1:50, Invitrogen). M1 and M2 macrophages were stained with rat anti-CD86 and anti-CD206 monoclonal antibodies (1:200, Invitrogen), respectively. Alexa Fluor 555-labeled donkey anti-rat IgG (1:1000, Abcam) was used as the secondary antibody, and cell nuclei were stained with DAPI. The immunofluorescence staining procedure was previously described [48 (link),51 ].

Immunofluorescence staining was performed to identify macrophages and their subtypes. Rat anti-F4/80 monoclonal antibodies (1:50, Invitrogen) were used to stain total macrophages. Dual staining of the macrophage M1/M2 subtypes was conducted with rat anti-mouse CD86 monoclonal antibodies (1:200, Invitrogen) and rabbit anti-mouse CD206 antibodies (1:500, Abcam). RNA was isolated from the harvested skin tissues surrounding the wound site using TRIzol reagent (Invitrogen). Macrophage polarization in the tissues was also quantified by determining the ARG1/TNF-α gene expression ratio as in the cell culture. For western blot analysis, tissues around the wound site were harvested, weighed, and sliced into pieces. Proteins were extracted and assayed and then separated by SDS‒PAGE as previously described [51 ]. The protein bands (n = 6) for TNF-α, ARG1, and β-actin were scanned and quantified with ImageJ software. Protein expression values were normalized to the β-actin levels.