Goat anti rabbit igg af555

iDISCO was carried out as previously described (http://www.idisco.info)^{22 (link)}. The following antibodies were used: goat anti-mouse VEGFR3 (R&D, No. AF743,1:400), rat anti-mouse LYVE1 (R&D, MAB2125,1:400), rabbit anti-mouse LYVE1 (AngioBio, No. 11-034,1:200), mouse anti-human VEGFR3 (Santa Cruz Biotechnology, SC-28297, 1:200), rabbit anti-human LYVE1 (Angio-Proteomie, 102-PA50S, 1:200), goat anti-mouse IgG–AF647 (Invitrogen, A21235, 1:500), donkey anti-goat IgG–AF647 (Invitrogen, A21447, 1:500), goat anti-rabbit IgG–AF555 (Invitrogen, A21428, 1:500). Subsequently, the transparent optic nerves with optic nerve sheaths were imaged using a Leica confocal microscope (Stellaris 8). Three-dimensional rendering was completed using Imaris 8 software (Oxford Instruments).

5×10⁴ isolated PMNs seeded on poly-L-lysine-coated glass coverslips (BD Biosciences) in tissue-culture wells and allowed to settle prior to stimulation as described above. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-MPO (Dako) and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma) and NETs were visualized using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss).

The 5 × 10⁴ isolated PMNs were seeded on poly-L-lysine-coated glass coverslips (BD Biosciences, San Jose, CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-NE (Abcam, Cambridge, MA, USA), rabbit anti-MPO (Dako, Glostrup, Denmark), two different rabbit anti-PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse anti-histone H1 + core proteins (EMD Millipore, Billerica, MA, USA), and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen Life Technologies, San Diego, CA, USA), and goat anti-mouse IgG AF647. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 fields (at least 1,000 PMNs) per case was evaluated for MPO/NE and DNA co-staining; nuclear phenotypes and NETs were counted and expressed as percentage of the total number of cells in the fields.