Fig 1: Cryoloading introduces alien compounds into the nerve terminal. (A) Fresh chick brain synaptosomes were plated on coverslips, fixed and immunostained for SV2 (DSHB, 1:1), a SV marker, and either the NMDA receptor subunit, NR2B (Abcam, ab65783; 1:200; upper panel) or GABA-A α1 receptor (Millipore 06-868 1:200, lower panel), as labeled. Staining overlays are shown on the right. SV2 stains SVs selectively and fills the presynaptic terminal. NMDA and GABA-A receptor antibodies identify small patches on the SSM surface (*, enlarged inset), marking the scab of postsynaptic membrane (see also Figure 2A). Light microscopy was carried out on a Zeiss Axioplan upright microscope with a 63X oil immersion, 1.45 NA objective. Image stacks used for fixed samples, as in A, were deblurred by iterative deconvolution using the Zeiss turnkey software, as described (Li et al., 2004). (B) Nomarski bright field (left), FM4-64 uptake (2 mM Ca2+/40 mM K+; center) and overlay (right) identifying terminals with functional SV recycling in the SSM fraction. FM dye stained SSM images were background corrected using the Zeiss software function. (C) Chick synaptosomes cryoloaded with 3 kDa dextran-FITC with the marker at the indicated concentrations in the freezing buffer imaged by bright field (upper panel) and fluorescence microscopy (lower panel).
Fig 2: Embryonic chicken forebrain neurons can be grown in vitro and GluN2B is expressed in chicken forebrain tissue in the fetal period. Chicken forebrain was harvested at E10 and the cell culture was incubated overnight, before inspected at DIV1, using: A, Light microscopy and B, Immunostaining with neuronal marker NeuN and nuclear stain DAPI. B1: Brightfield image. B2: Staining with DNA marker DAPI, visualized by UV light. B3: Immunostaining with NeuN, visualized by fluorescence microscopy. B4: Composite image of DAPI stain and NeuN immunostaining. C: Western blot stained with anti‐GluN2B antibody, concentration 1:1000 (ab65783, Abcam, UK) and anti‐β‐actin antibody. Lanes: 1‐3: HEK cells transfected with GluN2A, GluN2B or CMV plasmid, respectively. Lane 4: Homogenized chicken forebrain tissue harvested at E10. Lane 5: Chicken embryo forebrain cell culture, harvested at E10 and analyzed at DIV1. Lane 6: Homogenized mouse cerebellum harvested at P21. D: Time series of GluN2B protein expression in homogenized chicken embryo forebrains, from E7 to E18. GluN2B protein expression relative to internal control protein β‐actin expression. The values are normalized to expression level at E7 (n = 3), and statistical significance was investigated using the Kruskal‐Wallis test. Variation is given as standard deviation and a representative example of western blot of GluN2B and β‐actin is shown below the graph
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