Alexa 488 conjugated donkey anti mouse igg

Selected sections at the level of the dDG were processed using the MOM kit as described above. Sections were incubated overnight at RT in a solution containing rabbit anti-GFP (1:2000; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and mouse anti-GAD65 (1:100; Millipore) or mouse anti-GFP (1:100; Invitrogen), guinea pig anti-VGLUT2 (1:5000; Millipore) and rabbit anti-VGAT (1:1000; Synaptic System) diluted in MOM diluent. After several rinses in KPBS, they were incubated for 2 h in Alexa488-conjugated donkey anti-rabbit IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-mouse (1:100; Jackson ImmunoResearch Laboratories, Inc.) or Alexa488-conjugated donkey anti-mouse IgG (1:200; Invitrogen), Cy5-conjugated donkey anti-guinea pig (1:100; Jackson ImmunoResearch Laboratories, Inc.), and Cy3-conjugated donkey anti-rabbit (1:100; Jackson ImmunoResearch Laboratories, Inc.) diluted in MOM diluent. After several rinses in KPBS, all sections were then mounted on superfrost-coated slides, dried overnight at RT and coverslipped with Fluoromount. The specimens were analyzed with confocal microscope (Zeiss).

Rat hippocampal neurons were cultured on poly-L-lysine-coated coverslips. After treatment with NMDA for 4 h, the cells were fixed with 4% (w/v) paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.1) for 1.5 h at 4°C, permeabilized with blocking buffer (0.1 M sodium phosphate buffer containing 0.4% (v/v) Triton X-100, 2% (v/v) donkey serum, and 1% (w/v) bovine serum albumin) for 30 min at room temperature, and then labeled with anti-drebrin A/E (C-term) and anti-NeuN antibodies at 4°C overnight. Alexa 488-conjugated donkey anti-mouse IgG (Invitrogen) and Alexa 647-conjugated donkey anti-rabbit IgG (Invitrogen) were used as secondary antibodies. F-actin was stained with Alexa 555-conjugated phalloidin (Invitrogen). For quantitative analyses of the immunofluorescence data, images of 100 randomly selected NeuN-positive neurons were collected using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) with a 100× oil immersion objective, and the signal intensities of the circular areas (40 μm in diameter) covering both the cell soma and the apical dendrites were measured using Image J analysis software.

Mice were anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in PBS. The brains were harvested, postfixed in 4% paraformaldehyde overnight at 4 °C, and dehydrated in 30% sucrose in PBS for cryoprotection before being sliced at a thickness of 15 μm on a freezing microtome (Leica, Germany) and mounted onto microscope slides. The sections were incubated with 0.3% Triton X-100 in PBS for 10 min, blocked with 10% donkey serum for 30 min, and then washed with PBS. For double immunofluorescence staining, the sections were incubated with a mixture of anti-MD2 (Abcam, ab24182, 1:100, rabbit) and anti-NeuN (Millipore, MAB377, 1:200, mouse), anti-Iba1 (Abcam, ab5076, 1:200, goat) or anti-GFAP (Abcam, ab4674, 1:200, chicken) antibodies overnight at 4 °C. After three rinses, the sections were incubated with a mixture of Alexa 594-conjugated donkey–anti-rabbit IgG (Invitrogen, 1:400) and Alexa 488-conjugated donkey–anti-mouse IgG (Invitrogen, 1:400), Alexa 488-conjugated donkey–anti-goat IgG (Invitrogen, 1:400), or Alexa 488-conjugated donkey–anti-chicken IgG (Invitrogen, 1:400) antibodies for 2 h at room temperature. The slides were washed and cover-slipped with mounting media containing DAPI (Abcam, ab104139) for observation under a confocal laser scanning microscope (FV10, Olympus, Japan).