Alexa 488 conjugated goat antirat igg

Immunocytochemistry was performed as described previously [21 (link)]. For detection of HA-tagged proteins, we used rat monoclonal anti-HA antibody (clone 3F10; 1:1,000; Roche) and Alexa488-conjugated goat anti-rat IgG (Thermo Fisher Scientific) as the primary and secondary antibodies, respectively. Fluorescence was observed with a laser-scanning confocal fluorescence microscope (FV10i-DOC, Olympus).

Immunostaining of the resting and stimulated RBL-2H3 cells was performed as previously described36 (link). In some cases, disruption of the F-actin network was performed by incubation of cells in medium containing 1 μM LatB (Sigma-Aldrich) for 15 min prior to antigen stimulation or fixation. For staining of F-actin, the cells were incubated with 5 U/ml BODIPY FL PhallAcidin (Thermo Fischer Scientific) in PBS for 1 h after fixation, permeabilisation, and blocking. For immunostaining, Alexa488-conjugated goat anti-rat IgG and Alexa594-conjugated goat anti-mouse IgG antibodies (Thermo Fischer Scientific) were used as secondary antibodies. Stained cells were irradiated with a blue-beam (488 nm) from Argon ion laser and with a green-beam (543 nm) from He/Ne laser, and fluorescent images were acquired using a LSM510 meta/ConfoCor2 system (Carl Zeiss) equipped with the ×63 oil/1.4 NA (Fig. 1B, −Ag) and ×40 water/1.2 NA objectives (others), and with the FITC/Rhodamine (505–530 nm, > 560 nm) filter set. Co-localisation analyses were performed using Image J (Fiji). All images shown in this study are representative of at least 10 stained cells.

A mouse monoclonal antibody against GM2 ganglioside (GMB28; immunoglobulin M, 1:20) was kindly donated by Dr. Tai (Department of Tumor Immunity, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). An anti-GFAP (Dako, Carpinteria, CA, USA, 1:1000), anti-Iba1 (Wako Pure Chemical Industries, Osaka, Japan, 1:500), anti-CD68 (clone FA-11, AbD Serotec Ltd., Oxford, UK, 1:100), anti-NeuN (EMD Millipore, Billerica, MA, 1;1000) and anti-S100ß (GeneTex, Irvine, CA, 1:100) were used as primary antibodies. As secondary antibodies, Alexa-488-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgG (1:1000), Alexa-568-conjugated goat anti-mouse IgM (1:1000), Alexa-488-conjugated goat anti-rat IgG (1:1000), Alexa-488-conjugated goat anti-rabbit IgG (1:1000), Alexa-568-conjugated goat anti-rabbit IgG (1:1000) (all purchased from Molecular Probes, Eugene, OR, USA), and Histofine Simple Stain MAX-PO(R) (Nichirei Co., Tokyo, Japan) were used.

YAC128 NSCs were transferred to 12 mm round cover slips coated with PLO/Laminin in a four‐well plate (BD Biosciences). The cells were then washed with chilled PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Next, the cells were permeabilized and blocked with 0.1% Triton X‐100 (Sigma‐Aldrich) and 5% normal horse serum (Vector Laboratories) diluted in PBS for 30 minutes at room temperature. The cells were then incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody. The following secondary antibodies were used: Alexa‐555‐conjugated goat anti‐mouse IgG; Alexa‐555‐conjugated goat anti‐rabbit IgG; Alexa‐488‐conjugated goat anti‐rat IgG; and Alexa‐488‐conjugated goat anti‐rabbit IgG (1:200, Molecular Probes). Images were obtained using two confocal laser‐scanning microscope systems: a Zeiss LSM 880 scanning confocal microscope (Zeiss) and a Leica TCS Sp5 II confocal microscope (Leica). The following primary antibodies were used: anti‐NESTIN (1:40, Developmental Studies Hybridoma Bank), anti‐SOX2 (1:200, Millipore) for the detection of uncommitted neural stem cells, anti‐MAP2 (1:500, Millipore), anti‐Tuj1 (1:500, Millipore) for the detection of differentiated neurons, anti‐HTT (1:100, Sigma‐Aldrich) and anti‐ubiquitin (1:50, Santa Cruz Biotechnology).