Donkey anti rabbit 647

The primary antibodies used in this study were rabbit polyclonal NE (Abcam, Cambridge, MA, 1:100), fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal to MPO (Abcam, 1:200), rabbit polyclonal citrulline histone 3 (H3Cit; Abcam, 1:100), rat monoclonal CD11b (Abcam, 1:100), and rat monoclonal Ly6G (Abcam, 1:100). Secondary antibodies against the primary antibodies were donkey anti-rabbit AlexaFluor 568 (Invitrogen, Grand Island NY, NY, 1:500), donkey anti-rabbit 647 (Abcam, 1:200), donkey anti-rat 488 (Abcam, 1:500), and donkey anti-rat AlexaFluor 568 (Abcam, 1:500), respectively. Matched isotype controls were used as internal controls.

Free-floating sections were washed 3 times with PBS (15 min each), blocked for 1 h in blocking buffer (2.5% BSA, 0.3% Triton X-100 PBS), and incubated overnight at 4 °C with the primary antibody in the blocking buffer. The following antisera were used: rabbit anti-AQ-4 (1:500, #AB3594; Millipore, Sigma Aldrich, St. Louis, MO, USA), rabbit anti-YKL-40 (1:300, #PA5-43746; ThermoFisher Scientific, Waltham, MA, USA), goat anti-CD31 (1:500, #AF3628; R&D Systems, Minneapolis, MN, USA), rabbit anti-NeuN (1:1000, #ABN78; Millipore, Sigma-Aldrich, St. Louis, MO, USA), and mouse anti-glial fibrillary acidic protein (GFAP; 1:500, #G3893; Sigma-Aldrich, St. Louis, MO, USA). The next day sections were washed 3 times with the blocking buffer (15 min each), and subsequently incubated for 1 h at room temperature with the secondary antibody in the blocking buffer. The secondary antibodies consisted of donkey anti-rabbit 647 (1:1000, #AB150075; ABCAM, Cambridge, MA, USA), donkey anti-mouse 594, anti-goat 488, and anti-rabbit 488 (each 1:1000, #A21203, #A11055, #A21206, respectively; Invitrogen, and ThermoFisher Scientific, Waltham, MA, USA). Lastly, sections were washed 2 times with PBS (10 min each), and finally mounted onto slides covered with DAPI mounting media (#F6057; Sigma-Aldrich, St. Louis, MO, USA).