i3N iPSCs or i3Neurons in coverslips were fixed with conditioned medium containing 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated for 1 hr in blocking solution containing PBS, 0.01% Triton X-100, and 5% normal goat serum. The cells were then incubated in blocking solution containing primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 hr. Images were acquired with an LSM880 confocal system (Zeiss) with Airyscan and a 20× or 63× oil-immersion objective lens. Antibodies used for immunocytochemistry were those against SOX2 (sc-17320S; Santa Cruz Biotechnology), OCT4 (sc-5279; Santa Cruz Biotechnology), TRA-1-81 (sc-21706; Santa Cruz Biotechnology), MAP2 (AB5622 or MAB3418; Millipore), VGlut1 (MAB5502; Millipore), βIII tubulin (TUJ1; Aves Labs), neuronal nuclear antigen (MAB377; Millipore), GABA (A2052; Sigma), HT7 (MN1000; Thermo Fisher), ankyrin G (N106/36; NeuroMa), synapsin-1 (D12G5; Cell Signaling), Olig2 (AB9610; Millipore), GFAP (MAB3402; Millipore), and GluR2/3 (AB1506; Millipore).
Mab5502
Manufactured by Merck Group
Sourced in Germany, France
MAB5502 is a laboratory equipment product from Merck Group. It is a high-performance instrument used for various analytical applications. The core function of MAB5502 is to perform precise and reliable measurements, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab9610, by Merck Group (2 mentions)
A2052, by Merck Group (1 mentions)
Mab377, by Merck Group (1 mentions)
Sc 5279, by Santa Cruz Biotechnology (1 mentions)
Mab3402, by Merck Group (1 mentions)
Sc 21706, by Santa Cruz Biotechnology (1 mentions)
Ab1506, by Merck Group (1 mentions)
Ab5622, by Merck Group (1 mentions)
Lsm 880 confocal system, by Zeiss (1 mentions)
Mab3418, by Merck Group (1 mentions)
Ab9361, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Ab702, by Abcam (1 mentions)
Sliding microtome, by Leica camera (1 mentions)
Ab7260, by Abcam (1 mentions)
Mab1572, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
N4142, by Merck Group (1 mentions)
Ab144p, by Merck Group (1 mentions)
Ab1778, by Merck Group (1 mentions)
5 protocols using mab5502
1
Immunocytochemistry of iPSCs and Neurons
2
Immunostaining Protocol for Spinal Cord and Neuromuscular Junctions
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C5–C7 spinal segments or musculocutaneous nerves were cut into 15-µm-thick frozen sections for immunostaining. The blocking buffer was composed of 5% goat serum and 3% bovine serum albumin diluted in 0.1 M phosphate buffer saline (PBS). Signal was detected with Alexa fluor 546 or 488 coupled secondary antibodies (1:1000, Invitrogen). Primary antibodies were: goat anti- choline acetyltransferase (ChAT, 1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Calretinin (1:300, ab702, Abcam), mouse anti-Parvalbumin (1:1000, Mab1572, Millipore), rabbit anti-CAMKII (1:500, ab104224, Abcam), rabbit anti-vesicular GABA transporter (VGAT; 1:800, NO131013, Synaptic Systems), mouse anti-vesicular glutamate transporter 1 (vGlut1; 1:1000, Mab5502, Millipore), rat anti-major histocompatibility complex 1 (MHC1; 1:300, sc-59199, Santa Cruz), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, AB7260, Abcam), rabbit anti-Iba1(1:1000, 019–19,741, Wako), and rabbit anti-Oligo2 (1:500, ab9610, Merck Millipore).
On day 50 after BPA, the biceps were collected and 7-µm horizontal sections were prepared with a sliding microtome (Leica, Germany) and double stained with rabbit anti-NF200 (1:500, n4142, Sigma) and α-BT (1:1000, Molecular probes, USA) to visualize neuromuscular junctions (NMJs).
On day 50 after BPA, the biceps were collected and 7-µm horizontal sections were prepared with a sliding microtome (Leica, Germany) and double stained with rabbit anti-NF200 (1:500, n4142, Sigma) and α-BT (1:1000, Molecular probes, USA) to visualize neuromuscular junctions (NMJs).
3
Immunohistochemical Characterization of Neurons
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The normal donkey serum (NDS), the 4′,6′-diamidino-2-phenylindole (DAPI), and the anti-calbindin antibody produced in mouse (C9848) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The anti-glutamate receptor delta 2 (GluRδ2) antibody developed in goat was obtained from Santa Cruz (Le Perray en Yvelines, France). The anti-calbindin (AB1778), anti-VGlut1 (MAB5502), and anti-VGlut2 (AB2251) antibodies, produced respectively in rabbit, mouse, and guinea pig, as well as Cy3-conjugated donkey anti-guinea pig (DAGp) were from Millipore (Molsheim, France). Alexa 488-, Alexa 568-, or Alexa 546-conjugated donkey anti-goat (DAG), anti-mouse (DAM), or anti-rabbit (DAR) secondary antibodies were purchased from Invitrogen (Cergy Pontoise, France). Recombinant human PTN expressed in Spodoptera frugiperda insect cells came from Calbiochem (VWR, Strasbourg, France). Drugs used for electrophysiological recording were from Tocris Bioscience (Bristol, UK) or Ascent Scientific (Weston-Super-Mare, UK).
4
Rat Neuron Culture and Synaptic Analysis
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Neurons were cultured from embryonic day 18 rat embryos as described previously (Phillips et al., 2003; Fernández-Monreal et al., 2009) and grown for 12 or 21 days. In some cases, neurons were transfected at day 15 with pEGFP-n1 to fill dendrites with GFP and grown for an additional 7 days as described (Fernández-Monreal et al., 2009) . For synaptic measurements, neurons were fixed and immunolabeled with an affinity purified antibody raised against the Pcdh-g constant cytoplasmic domain (Phillips et al., 2003) . This antibody was validated in (Fernández-Monreal et al., 2010) by showing that it labels the same puncta as labeled with a commercially available monoclonal antibody to the Pcdh-g constant domain (clone 195/5, UC Davis/NIH NeuroMab Facility). For synaptic labeling, neurons were colabeled with goat anti-PSD-95 (Abcam Cat# ab12093, RRID:AB_298846) and mouse anti-vGlut (Millipore Cat# MAB5502, RRID:AB_262185). For spine measurements, GFP filled neurons were stained with anti-Pcdh-g only.
5
Immunostaining of Brain Tissue
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Tissue immunostaining was carried out as previously described (Szczesna et al., 2014) . The following primary antibodies were used: Ms-Gsk3b (ab93926; Abcam), Rb-Gsk3b pTyr216 (ab75745; Abcam), Rb-mAbGsk3b pSer9 (9323S; Cell Signaling), Rb-Nfkb p65 (6956; Cell Signaling), Rb-CD11b (Ab133357; Abcam), Ms-Vglut1 (MAB5502; Millipore), Rb-Psd95 (2507; Cell Signaling), and Rb-Bdnf (ab108319; Abcam). Specific secondary antibodies for each condition were used (Alexa Fluor 555 Rb-IgG, A21429, Alexa Fluor 488 Ms-IgG, A21200; Life Technologies). All pictures were obtained using a Zeiss microscope at 403 magnification and processed using ImageJ Fiji version 1.50g. Tile images from sagittal brain sections were obtained at 203 magnification using a Zeiss microscope. Z project and Tile were carried out in order to get complete staining with all antibodies in a larger area. Densitometric analysis was obtained as previously described (Szczesna et al., 2014) .
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