Fitc donkey anti rat

Immunostaining of cultures was performed as described (Ma et al., 2008b (link)). Neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After permeabilization with 0.20% Triton X-100 in blocking buffer (1% bovine serum albumin [BSA], 5% normal goat and/or donkey serum in PBS, pH 7.4), followed by incubation for 50 min in blocking buffer at room temperature, cells were doubly or triply stained with appropriate primary antibodies overnight at 4°C. Primary antibodies were visualized with appropriate secondary antibodies: Cy3-donkey anti-mouse, fluorescein isothiocyanate (FITC)–donkey anti-rabbit, FITC-donkey anti-rat, and FITC-donkey anti-guinea pig immunoglobulin G (IgG) were from Jackson ImmunoResearch Laboratories (West Grove, PA); Alexa Fluor 633–goat anti-rabbit and anti-mouse IgG were from Molecular Probes (Eugene, OR). Images were obtained using a Zeiss (Jena, Germany) LSM510 confocal microscope. Hoechst nuclear stain (Invitrogen, Grand Island, NY) and tetramethylrhodamine isothiocyanate (TRITC)–phalloidin (Sigma-Aldrich, St. Louis, MO) were used where indicated.