Goat anti human mouse mpo

Formalin-fixed paraffin embedded sections (5 μm thickness) were baked at 60°C for 20 minutes, dewaxed in xylene for 1 hour, and rehydrated through an alcohol series. Antigen retrieval with 1X Reveal Decloaker (Biocare Medical) was performed using the capillary gapping method with a vegetable steamer for 35 minutes. Once sections were cooled and washed, they were blocked in Background Sniper (Biocare Medical) for 10 minutes. Primary antibodies, Chicken anti-Mouse/Human/Rat GFAP (Invitrogen PA1–10004) and Goat anti-Human/Mouse MPO (R&D AF3667) were diluted to 1 μl/mL and 0.5 μl/mL, respectively, in sniper and added to sections overnight at 4°C. Primaries were removed, and sections were washed in TBST twice before species-specific secondary antibodies Donkey anti-Chicken Ax594 at a 2 μl/mL concentration and Donkey anti-Goat IgG Ax488 at a 4 μl/mL concentration were added for 2 hours at room temperature. A TBS with 0.1% Triton X wash and distilled water wash performed, and then coverslips were mounted onto sections with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories) before imagining. Sections were imaged at 20x or 40x on an Olympus Fluoview FV1000 and neutrophils were counted on a Zeiss Axio Imager.M1 (20x and 40x objective).

Frozen sections of aortic root were fixed with 4% PFA for 10 min, followed by permeabilization in 0.2% Triton X-100 for 8 min. The sections were blocked by 1% BSA and 1% donkey serum in PBS for 30 min. Sections were then stained with primary Abs: rabbit anti-citrullinated histone 3 (Abcam, ab 5103), rat anti-mouse Ly-6G (BioLegend), rat anti-mouse F4/80 (BioLegend), biotin conjugated hamster anti-mouse MCP-1 (BioLegend), goat anti-human/mouse MPO (R&D), hamster anti-mouse IL-1β (BioLegend), followed by staining with Alexa Fluor 488-conjugated donkey anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-goat IgG, Alexa Fluor 555-conjugated donkey anti-rabbit IgG and Alexa Fluor 568-conjugated donkey anti-hamster IgG, Alexa Fluor 647-conjugated donkey anti-rabbit IgG, or Alexa Fluor 594-conjugated donkey anti-rat IgG (all from Invitrogen) (Life Technologies). Cells and tissue sections were counterstained with DAPI (Life Technologies) before being mounted in ProLong Gold (Life Technologies) and examined by confocal microscopy.