Alexa 488 conjugated donkey anti mouse secondary antibody

Neuronal nuclear protein (NeuN) immunostainings were performed on resected human hippocampal tissue derived from control and mTLE individuals. Sections were fixed for 10 min in 4% PFA, washed in 1× PBS and blocked in 3% Normal Goat Serum, 0.2% Triton in 1× PBS, pH 7.4, for 1 h at RT. Incubation was performed with mouse anti-NeuN antibody (ab104224, Abcam) in blocking solution overnight at 4°C. After washing, sections were incubated with Alexa-488 conjugated donkey anti-mouse secondary antibody (A21202, Thermo Fisher Scientific) for 1.5 h at RT, followed by three washes in 1× PBS and DAPI staining. Mounting was performed using Vectashield (Vector Labs) and images were acquired with a brightfield microscope (Axio Scope A1, Zeiss).

Cells were washed once with FACS buffer and stained with antibodies for 30 min at 4°C. Samples were washed once with FACS buffer and analyzed on MACSQuant VYB (Miltenyi Biotech). The results were analyzed using Flowjo v10 (FlowJo, LLC). Fluorochrome conjugated human antibodies are listed in Table S2. FACS analysis of CD47 was done using anti-CD47 antibody (Bio-Rad, MCA911, 1:25) and Alexa 488 conjugated donkey anti-mouse secondary antibody (Thermo Fisher Scientific). PI solution (Miltenyi Biotec, 130-093-233, 1:100) was also used in specific flow cytometry analysis.

Cells were plated on glass coverslips. For the internalization assay, cells were serum-starved for 4 h and incubated with Alexa555-EGF (40 ng/mL) and/or 13A9 antibody (20 μg/mL) for 1 h at 4 °C and shifted to 37 °C for various timepoints to allow internalization. The cells were then fixed in 4% paraformaldehyde (PFA) in PIPES buffer (0.2% BSA, 0.1% Triton X-100, and 1× PBS) for 10 min at room temperature. PFA-fixed cells were permeabilized with 0.1% Triton X-100 in 1× PBS for 5 min at room temperature. The cells were then incubated with 2% BSA in 1× PBS for 30 min at room temperature, followed by incubation for 30 min with Alexa488-conjugated donkey anti-mouse secondary antibody (A21202, Thermo Fisher Scientific, Waltham, MA, USA). The nuclei were DAPI-stained. The coverslips were analyzed using a confocal microscope (CLSM TCS SP8 STED, Leica), and the images were further processed with the ImageJ software.