Alexa 488 conjugated donkey anti goat igg

Tumor samples were fixed in 10% formalin and were embedded in paraffin, which was cut into 5-µm-thick sections. For immunohistochemical staining, deparaffinized sections were blocked with 10% normal horse serum for 1 h and then incubated with primary antibodies against TGF-β, CD-31 and IL-17A (1∶100). The samples were incubated with biotinylated secondary antibody (DAKO code K0675; DAKO Corp., Carpinteria, CA) and peroxidase-labeled streptavidin (DAKO code K0675). Staining was developed using the 3-3 diaminobenzidine (DAB) substrate chromogen system (DAKO Corp.).
To immunofluorescence staining, deparaffinized sections were blocked with 10% normal horse serum for 1 h and then incubated with primary antibodies against TGF-β and IL-6. And then, the samples were incubated with Alexa 594-conjugated donkey anti-rabbit IgG (Invitrogen).
Co-immunofluorescence staining was also performed to assess co-localization of CD4 and Ror-γ. The sections were incubated overnight with a mixture of anti-CD4 and anti-Ror-γ in 4°C followed by washes with PBS and incubation with a mixture of Alexa 488-conjugated donkey anti-goat IgG and Alexa 594-conjugated goat anti-rat IgG (Invitrogen). The reactions were examined using an immunofluorescence microscope.

After recordings, slices were processed for simultaneous detection of the biocytin-filled neurons and GFP-labeled axon fibers and terminals in order to identify the recorded cells and evaluate the efficiency of the transfection, respectively. Slices were fixed overnight at 4 °C in a solution containing 4% PFA in PB. Then they were rinsed in PB, cryoprotected in 20% sucrose and quickly frozen on dry ice. After several rinses in KPBS, slices were incubated in a solution containing normal donkey serum (NDS, 1:30; Vector Laboratory) diluted in KPBS with 0.3% Triton- X100, for 2 h at RT. They were incubated in a solution containing goat anti-biotin (1:200) and rabbit anti-GFP (1:2000), diluted in KPBS containing 0.3% Triton-X100 and NDS (1:100), overnight at RT. After several rinses in KPBS, slices were incubated for 2 h in Alexa488-conjugated donkey anti-goat IgG (1:200; Invitrogen), and Cy3-donkey anti-rabbit IgG (1:100) diluted in KPBS with 0.3% Triton-X100. After rinses in KPBS, slices were mounted on slides and coverslipped with Fluoromount. The specimens were analyzed with a fluorescence microscope (Nikon 50i) or confocal microscope (Zeiss).

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).

Primary antibodies used were monoclonal anti-synaptobrevin 2 (104211 from Synaptic system; 1:5,000); monoclonal α-tubulin (T5168 from Sigma; 1:5,000); rabbit polyclonal anti-CgA (EL-35)^{23 (link)} (1:500 for IF, 1:1,000 for Western blotting); goat polyclonal anti-CgA (sc-23556 from Santa Cruz Biotechnology inc; 1: 200); rabbit polyclonal anti-Myo1b (HPA 013607 from Sigma prestige antibodies; 1:200 for IF, 1:250 for Western blotting); mouse monoclonal anti-GM130 (610822 from BDBiosciences; 1:1,000); rabbit polyclonal anti-furin (Ab3467 from Abcam; 1:200); sheep polyclonal anti-human TGN46 (AHP500 from AbD serotec; 1:500); rabbit polyclonal anti-p34-Arc/ARPC2 (07-227 from Millipore; 1:400); goat anti-type III collagen (1330-01 from Southern Biotech; 1: 200). For IF, secondary antibodies used were Alexa 488-conjugated donkey anti-rabbit IgG; Alexa 594-conjugated donkey anti-rabbit IgG; Alexa 647-conjugated donkey anti-rabbit IgG; Alexa 488-conjugated donkey anti-mouse IgG; Alexa 488-conjugated donkey anti-goat IgG; Alexa 594-conjugated donkey anti-goat IgG; Alexa 594-conjugated donkey anti-sheep IgG; Alexa 647-conjugated donkey anti-mouse IgG (Invitrogen; 1:500). For Western blotting, anti-rabbit, anti-mouse and anti-goat secondary antibodies conjugated to horseradish peroxydase (Santa Cruz biotechnologies; 1:2,000) were used.

Primary antibodies used for rodent experiments were rabbit anti-Caspr (Abcam, UK), mouse CC1 monoclonal antibody (Abcam, UK), rabbit anti-GAPDH (Sigma, UK), mouse anti-myelin-associated glycoprotein (MAG, Millipore, UK), rabbit anti-Kv1.1 (Abcam, UK), rat anti-myelin basic protein (MBP, Serotec, UK), chicken anti-neurofilament heavy chain (NFH, Encor Biotechnologies, Florida, United States), rabbit anti-Olig2 (Sigma, UK), mouse anti-O4 (Immunosolv, UK), mouse anti-NG2 (Millipore, UK), mouse anti-proteolipid protein (PLP, Millipore, UK), goat anti-Scribble (C20, Santa Cruz Biotechnology, US), mouse anti-pan sodium channel (Sigma, UK). Secondary antibodies used were Alexa 488-conjugated goat anti-rabbit IgG, Alexa 488-conjugated donkey anti-goat IgG, Alexa 568-conjugated donkey anti-rat IgG, Alexa 568-conjugated donkey anti-mouse IgG, and Alexa 568-conjugated goat anti-rabbit IgG (all from Invitrogen, UK), DyLight 405-conjugated donkey anti-rat IgG, and DyLight 405-conjugated donkey anti-chicken IgG (Stratech, UK).

The primary antibodies used for immunocytochemistry in this study were rabbit anti-Aβ42 (1:35, Millipore), mouse anti-NES (nestin) (1:500, R&D), rabbit anti-OCT4 (1:500, Abcam), mouse anti-SYP (synaptophysin) (1:500, Abcam), mouse anti-TRA-1-81 (1:250, BD Pharmingen), and rabbit anti-TUBB3 (tubulin β3 class III) (1:1000, Covance). The secondary antibodies included Alexa594-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:200, Invitrogen), and Alexa488-conjugated donkey anti-mouse IgG (1:200, Invitrogen). The antibodies used for western blotting were mouse anti-GSK3β (glycogen synthase kinase 3β) (1:500, Cell Signaling Technology), mouse anti-phospho-GSK3β (Ser9) (1:500, Cell Signaling Technology), mouse anti-tau (1:500, Abcam), mouse anti-phospho-tau (Ser396) (1:1000, sigma), mouse anti-phospho-tau (Thr181) (1:500, Cell Signaling Technology), and mouse anti-TUBB3 (1:10000, Biolegend). The secondary antibodies used included goat anti-mouse IgG-HRP (horseradish peroxidase) (1:20000, GE Healthcare), goat anti-rabbit IgG-HRP (1:20000, GE Healthcare), and donkey anti-goat IgG-HRP (1:2500, Santa Cruz). The immune complexes were detected using an enhanced chemiluminescent substrate (ECL, Biotools).