Rabbit anti mpo

Brain sections were fixed in methanol and membranes were ruptured with 0.25% Triton‐X100. After blocking with 1% BSA, sections were incubated overnight at 4°C using the following primary antibodies: rabbit anti‐Iba‐1 (Wako), rat anti‐CD16 (BD biosciences), goat anti‐CD206 (R&D Systems), rat anti‐F4/80 (BioLegend), rabbit anti‐MPO (Abcam), rabbit anti‐MBP (Abcam), mouse anti‐NF200 (SigmaAldrich), mouse anti‐BrdU (GeneTex), and rabbit anti‐CNPase (Abcam). TUNEL assays were performed using in situ cell death detection kit, fluorescein (Roche). Positive cells from the peripheral areas of the infarct core were obtained from the microscope field, including the corpus callosum and striatum in the white matter area. Nine images were randomly selected for each region for calculation.

Three-μm-thick sections in paraffin of human PsA and OA synovia were stained after deparaffination in xilene (5 min, two times), followed by passages in: absolute ethanol (3 min), 95% ethanol in water (3 min), 80% ethanol in water (3 min), 70% ethanol in water, and antigen retrival (5 min at 95°C in 10 mM sodium citrate, pH 6.0). Slides were saturated with blocking buffer (PBS, 0.05% tween 20, 4% BSA) for 1 hour at room temperature. Specimens were stained with a polyclonal rabbit anti-LL37 (Innovagen), rabbit anti-MPO (Abcam), mouse anti-MxA (Novus Bio), monoclonal mouse anti-LL37 (Mab137), polyclonal rabbit anti-human C9 (ATLAS). The following antibodies were used: donkey anti-rabbit IgG AlexaFluor-568 or-647, anti-mouse AlexaFluor-647 and an anti-goat AlexaFluor-488 (Abcam). After washing, slides were mounted in Prolong Gold anti-fade media containing a DNA dye (DAPI) (Molecular Probes). CLSM observations were performed with a Leica TCS SP2 AOBS apparatus, using a 63x/1.40 NA oil objective. Acquisition of images was performed by a Leica confocal software 2.6 (Leica, Germany).

Mice were perfused under deep anesthesia with ice-cold PBS followed by perfusion with 10% formalin at 24 h after surgeries. The brains were removed and fixed in formalin at 4 °C overnight and dehydrated with 30% sucrose for 3 days. Brain samples were then snap-frozen at − 80 °C and cut into 10-μm-thick coronal sections using a cryostat (CM3050S; Leica Microsystems). Immunofluorescence staining was performed as previously described [36 (link)]. Briefly, brain samples were incubated overnight at 4 °C with primary antibodies including goat anti-Iba-1 (1:100, Abcam), goat anti-GFAP (1:100, Abcam), goat anti-NeuN (1:200, Abcam), rabbit anti-MC4R (1:500, Abcam), and rabbit anti-MPO (1:500, Abcam). The sections were then incubated with corresponding secondary antibodies (1:200, Jackson Immunoresearch, West Grove, PA) at room temperature for 2 h and followed by visualization using a fluorescence microscope (Leica Microsystems, Germany).

Lung tissue sections were fixed as reported above. Lung samples underwent antigen retrieval (pH 9 buffer) using a Dako PT Link pre-treatment module (Agilent, CA, USA). Samples were washed and blocked for 10 min (Dako protein block; Agilent, Santa Clara, CA) before being treated with primary antibodies overnight. Mouse anti-COL1A1, rabbit anti-fibronectin, mouse anti-ly6G, rabbit anti-MPO, rabbit anti-mannose receptor, rat anti-F4/80, and mouse anti-SMAD7 (Abcam, CAM, UK), and rabbit anti-OGG1 (Invitrogen, Carlsbad, CA) antibodies were used. Alexa Fluor 488-conjugated goat/anti-mouse and Alexa Fluor 647 goat/anti-rabbit (Invitrogen, CA, USA) were used as secondary antibodies. Glass cover slips were mounted with DAPI-containing fluoroshield (Abcam). Images were visualized using a Nikon Confocal Microscope and fluorescence was quantified using ImageJ software (Java 1.8.0_172).