Polymer horseradish peroxidase labeled goat anti rabbit igg

Back skin excised from the animals was fixed in 4% paraformaldehyde overnight and embedded in paraffin blocks. After sectioning, all samples were stained with hematoxylin and eosin (HE) to determine the inflammation infiltration. As for the HE dyeing, antibody rabbit anti-Ki-67 and polymer-horseradish peroxidase-labeled goat anti-rabbit IgG were used as the primary antibody and the secondary antibody (DAKO, Glostrup, Denmark), respectively. The positively stained epidermis was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Some tissue was routinely processed for paraffin sections. After deparaffinization and rehydration, immunohistochemistry was performed as described previously (Zou et al., 2015 (link)). The blocking solution was Dual Endogenous Enzyme Block (DAKO, Glostrup, Denmark). Rabbit anti-TWEAK, Fn14, Iba-1 or CD3 IgG (Abcam, Cambridge, MA, United States) and rabbit anti-epidermal growth factor receptor (EGFR, phosphorylated) IgG (Cell Signaling, Danvers, MA, United States) were used as primary antibodies (2 μg/ml). Polymer-horseradish peroxidase-labeled goat anti-rabbit IgG (DAKO) was secondary antibody (2 μg/ml). Brown-yellow color was developed by using 3, 3′-diaminobenzine-chromogen substrate (DAKO).
Some sections were stained with hematoxylin-eosin or Masson’s trichrome solution. The epidermal thickness was measured with H&E-stained sections. The number of appendage-like structures per mm² area was also counted on these sections. The wound area and histological evaluation were detailed in Supplementary Table S1.