Alexa 488 labeled goat antimouse igg1

Formalin‐fixed paraffin‐embedded tissue was stained as described previously (Mizee et al., 2014). In short, deparaffinization and antigen retrieval, sections were incubated overnight with appropriate antibodies (for details, see Table 2) and subsequently incubated with horseradish peroxidase EnVision kit (Dako, Denmark) followed by 3,3’diaminobenzidine‐tetrahydrochloride dihydrate (Dako, Denmark). All sections were counterstained with hematoxylin after which they were analyzed with a light microscope (AXIO Scope A1, Carl Zeiss, Germany). For cellular localization studies, sections were incubated overnight with appropriate antibodies followed by incubation with Alexa‐488‐labeled goat antimouse IgG1 (1:200; Molecular Probes, USA) and analyzed by confocal microscopy (Leica DMI 6000 SP8, Leica, Germany).

Neurons were fixed in 4% paraformaldehyde/4% sucrose solution for 15 min at 37 °C, permeabilized with 0.25% Triton X-100, and blocked with 0.5% fish skin gelatin at 7 and 16 DIV^{26 (link)}. The following antibodies were used: anti-SMI312 (BioLegend 837904, mouse IgG1/IgM, 0.5 μg mL⁻¹), anti-MAP2 [Abcam ab5392, chicken polyclonal IgY (IgG), 2.5 μg mL⁻¹], Alexa 488-labeled goat anti-mouse IgG1 (Molecular Probes A21121, 2 μg mL⁻¹), and Alexa 568-labeled goat anti-chicken IgG (Molecular Probes A11041, 8 μg mL⁻¹).