Mouse monoclonal anti nestin

The cultures were fixed in 4% paraformaldehyde in PBS for 1 hr at room temperature (RT) and incubated in icecold 20% methanol in PBS for 5 min at RT. Fixed specimens were permeabilized in 0.5% Triton X-100 in PBS overnight at 4 C, and block in 15% basal serum albumin (BSA; Sigma) in PBS for overnight at 4 C. Specimens were incubated in primary antibodies with 1:500 dilution for 72 hr at 4 C as follows: mouse monoclonal anti-Nestin (Biolegend), mouse monoclonal anti-βIII-tubulin (Tuj1; Biolegend), goat polyclonal anti-synaptic vesicle glycoprotein 2A (SV2A; Santa Cruz), rabbit polyclonal anti-tropomyosin receptor kinase B (TrkB; Santa Cruz), and mouse monoclonal anti-GATA binding protein 3 (Gata3; Biolegend). After 3 times of 5 min washing with PBS, appropriate secondary antibodies were used to conjugate the primary antibodies for 4 hr at RT with 1:1,000 dilution, including goat anti-mouse Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, donkey anti-goat Alexa Fluor 594, and donkey anti-rabbit Alexa Fluor 594 (all are from Molecular Probes). All antibodies were diluted in 5% BSA and 0.05% Tween-20 in PBS. All specimens were mounted in ProLong Diamond Anti-fade Mounting with DAPI (Molecular Probes) prior to observing by BX53 Upright Microscope (Olympus) and imaged by cellSens software (Olympus).