Cy3 conjugated goat anti mouse igg antibody

By triple immunofluorescence labelling, OCT sections were incubated for 24 h at 4 °C with the combination of three primary antibodies: goat anti-GlyRα3 polyclonal antibody (1:50, SC-17282, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-GlyRα1 polyclonal antibody (1:50, 146,003, Synaptic Systems, Germany), or mouse anti-gephyirn monoclonal antibody (1:200, 147,021, Synaptic Systems, Germany) or chicken anti-NeuN polyclonal antibody (NeuN is neuronal nuclear protein and it is specific for neurons) (1:200, ABN91, EMD Millipore Corp., MA, USA).
These incubations were followed by incubation with secondary antibodies DyLight 405-conjugated Affinity Pure goat anti-chicken IgY, Alexa Fluor488-conjugated Affinity Pure donkey anti-goat IgG, Cy3 conjugated goat anti-mouse IgG antibody (1:200, Jackson Immuno Research, West Grove, PA, USA), or Cy5 conjugated donkey anti-mouse IgG antibody (1:200, Jackson Immuno Research, West Grove, PA, USA).
The immunoreactivity (IR) of each section was examined. The images were captured using a Zeiss LSM 700 Confocal Microscope (Zeiss, Jena, Germany).

ARPE-19 cells were seeded onto 8-well slides (ibidi µ-slide, Gräfelfing, Germany) at a density of 20,000 cells/cm². After 8 h, the cells were infected with freshly purified T. gondii ME49 tachyzoites (2 × 10⁴ parasites per cm²). After 24 h, 10⁸ BODIPY-labelled urinary small EVs were added to each well and incubated for another 16 h. The specimens were fixed in freshly prepared 2% PFA (20 min, RT). Following washes in PBS, the cells were permeabilized in acetone:methanol (1:1) for 20 min at −20 °C. After washes in 0.025% Tween-20 in PBS, the infected cells were incubated overnight at 4 °C with a surface-antigen-1 mouse monoclonal antibody (anti-SAG-1, 1:100 in PBS) as described in [42 (link)]. Following several washes in PBS, the cells were incubated for 45 min with 1 mg/mL Cy3-conjugated goat anti-mouse IgG antibody (1:400 dilution, Jackson ImmunoResearch 115-165-062, Cambridgeshire, UK). Nuclear counterstaining was performed using Hoechst (1 µg/mL. Sigma). Thereafter, the slides were mounted in Fluoromount (Sigma), placed onto glass slides and imaged using a confocal microscope (Zeiss LSM 880, Feldbach, Switzerland).