Af647 donkey anti rabbit igg

Immunohistochemistry was performed on freshly formaldehyde-fixed paraffin-embedded cortex sections (4 μm) of at least two GRPs in technical triplicates. After de-paraffinization and rehydration, antigen retrieval was performed in either 10 mm Sodium Citrate (pH 6) or 10 mm Tris/1 mm EDTA (pH 9) at 96 °C for 30 min. After blocking in 5% BSA and Normal Donkey Serum, tissue sections were incubated in primary antibody (DDX4, 10 μg mL⁻¹, ab13840, Abcam; CD9, 1:200, 312102, BioLegend; MCAM, 1:100, AF932, R&D systems, RGS5, 1:50, MA5-25584, Invitrogen; CD31, 1:200, M0823, Dako; TAGLN, 1:100, MAB78861, R&D systems) overnight at 4 °C, followed by incubation with secondary antibody diluted 1:200 in blocking solution for 1 h at room temperature: AF488 donkey anti-goat IgG, AF594 donkey anti-mouse IgG, AF647 donkey anti-rabbit IgG (all from Thermo Fisher Scientific, A11055">A11055, A21203">A21203, A31573">A31573, respectively). Stained tissue sections were mounted in fluorescent mounting media (Dako Agilent) and imaged with Olympus IX81 fluorescence microscope (Carl Zeiss Meditec, Germany) or Nikon ultra fast widefiled microscope (Nikon, Germany). Acquired images were analyzed using Fiji/ImageJ software v2.0.

Stimulated neutrophils were collected after 6 hours and stained with Ly6G-FITC antibody (clone 1A8, BioLegend) for 20 minutes. Cells were washed and fixed with 4% PFA (BD Biosciences) overnight. To permeabilize fixed cells, 0.1% TritonX (Fisher Scientific) was used. Cells were incubated with 10% normal donkey serum (NDS, Jackson ImmunoResearch) for 1 hour before addition of primary antibodies: ALF-161 Armenian hamster anti-mouse IL-1α (Fisher Scientific), and rabbit anti-mouse CD63 (clone EPR21151, Ab-cam). Primary antibodies were incubated overnight at 4°C. Cells were washed twice with FACS buffer (PBS with 1% BSA and 2 mM EDTA). AF546 goat anti-hamster IgG (ThermoFisher Scientific) and AF647 donkey anti-rabbit IgG (ThermoFisher Scientific) secondary antibodies were added and incubated at room temperature for 30 minutes. 4 μL of cells were mixed with 4 μL of Vectashield® antifade mounting media with DAPI (Vector Laboratories) and plated on a coverslip. Imaging was done using an LSM700 confocal microscopy (Optical Biology Core, UCI, Leica LSM700) and analyzed with Zen software.