Anti mpo

The wounds, together with unwounded skin margins, were excised, fixed with 10% formaldehyde, and embedded with paraffin. The sections (4 μm thick) were then deparaffinized and rehydrated. Antigen retrieval was performed at 95°C by microwave in 0.01 mol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched by exposing to 3% H₂O₂. After blocking with 5% BSA in PBS, the sections were incubated with anti-CD68 (1 : 100, Thermo Fisher Scientific), anti-MPO (1 : 100, Thermo Fisher Scientific), anti-CD31 (1 : 200, Santa Cruz Biotechnology), anti-desmin (1 : 100, Thermo Fisher Scientific), and anti-collagen I (1 : 100, Thermo Fisher Scientific), respectively, followed by incubating with the corresponding HRP-conjugated secondary antibodies. The antigen-antibody complex was visualized with a Diaminobenzidine (DAB) kit. For evaluation of staining, the overview of the positive-signal density was scored semiquantitatively as 1 (absent), 2 (low), 3 (medium), 4 (strong), and 5 (very strong). The median of scores from three observers, who were blinded to the treatment, was used for comparisons.

10 μm-thick corneal frozen sections were post-fixed with 4% PFA for 10 minutes, rinsed, and blocked with PowerBlock (Biogenx, San Ramon, CA) for 1 hour. Subsequently, section were incubated with following primary antibodies: anti-Sirt6 and Acetyl-Histone H3 (Lys9) from Cell Signaling Technology (Beverly, MA); anti-Acetyl-Histone H3 (Lys56) from Abcam (Cambridge, MA); anti-MPO from Thermo Fisher scientific; anti-CD3 from eBioscience (San Diego, CA), anti-CD45, CD19 and CD31 from BD Bioscience (San Jose, CA); anti-Iba1 from Wako (Osaka, Japan); anti-keratin 12 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-loricrin from Biolegend (San Diego, CA). After washing, retinal sections were incubated with appropriate Alexa Fluor 488 or 594-labeled secondary antibodies at room temperature for 1 hour. Corneal sections were mounted with Fluoroshield™ with DAPI histology mounting medium (Sigma-Aldrich, St. Louis, MO) and images were taken by fluorescence microscope (Olympus, Waltham, MA).

For immunohistochemistry, 5 μm tissue sections were deparaffinised and stained on a Leica Bond III autostainer, or manually by retrieving antigens in TRS6 (Dako Cytomation) for 20 minutes in a microwave oven [12 (link)]. Primary antibodies used for incubation were rabbit polyclonal anti-MPO (myeloperoxidase) (Thermo Scientific, 1:150) and biotinylated chicken polyclonal antibody against the S100A8/A9 heterocomplex (antibody chicken anti-MRP8/14 (mouse, rat), catalogue number A 1098.2, Immundiagnostik AG, Bensheim, Germany). Next, we employed a signal amplification technique based on streptavidin, biotin, and peroxidase [7 (link)]. Mouse spleen served as positive control, whereas omission of the primary antibody in serial sections served as negative control. Staining was visualized by Bond Polymer Refine Detection (Leica) or Vectastain ABC kits (Vector labs). Slides were counterstained with hematoxylin (Vector labs).