Journal: Journal of Neuroscience Research

Article Title: Light microscopic and heterogeneity analysis of astrocytes in the common marmoset brain

doi: 10.1002/jnr.24967

Figure Lengend Snippet: List of primary antibodies used

Article Snippet: Anti‐GluA1 antibody produced in guinea pig , Peptide corresponding to AA 271‐285 of rat GluR1 , Alomone lab, # AGP‐009, Polyclonal antibody, RRID:AB_2340961 , 3.2 μg/ml/1:250.

Techniques: Concentration Assay, Produced, Recombinant, Purification, Derivative Assay, Transduction

Journal: Journal of Neuroscience Research

Article Title: Light microscopic and heterogeneity analysis of astrocytes in the common marmoset brain

doi: 10.1002/jnr.24967

Figure Lengend Snippet: Antibodies used and amino acid conservation between species

Article Snippet: Anti‐GluA1 antibody produced in guinea pig , Peptide corresponding to AA 271‐285 of rat GluR1 , Alomone lab, # AGP‐009, Polyclonal antibody, RRID:AB_2340961 , 3.2 μg/ml/1:250.

Techniques:

Journal: Journal of Neuroscience Research

Article Title: Light microscopic and heterogeneity analysis of astrocytes in the common marmoset brain

doi: 10.1002/jnr.24967

Figure Lengend Snippet: Molecular heterogeneity of astrocytes in the marmoset cerebellum. (A) Lower magnification images showing labeling for astrocytic proteins with respect to the molecular layer (ML), Purkinje cell layer (PCL), granule cell layer (GCL), and white matter (WM). Calbindin is used as a Purkinje cell marker. DAPI staining (blue) shows the nuclear staining of interneurons in the ML and granule cells in GCL. Scale bar: 100 μm. (B) Detailed images of ML and GCL stained with specific markers for Bergmann glia (BG) such as GluA1, Kirk4.1, and velate astrocytes (VA) such as AQP4. EAAT2 was expressed in both BGs and VAs with higher expression in VAs. Purkinje cell bodies are indicated with a white asterisk. Scale bar: 20 μm. bv, blood vessel

Article Snippet: Anti‐GluA1 antibody produced in guinea pig , Peptide corresponding to AA 271‐285 of rat GluR1 , Alomone lab, # AGP‐009, Polyclonal antibody, RRID:AB_2340961 , 3.2 μg/ml/1:250.

Techniques: Labeling, Marker, Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SYNPLA, a method to identify synapses displaying plasticity after learning

doi: 10.1073/pnas.1919911117

Figure Lengend Snippet: SYNPLA detects synapse formation and labels synapses. (A) Diagram of a general PLA reaction. Note that A and B must be sufficiently close to permit complementary binding and ligation of ABo1 and ABo2 and subsequent amplification to make a rolony. (B) Diagram of PLA targeting recombinant presynaptic myc-NRXN and postsynaptic HA-NLGN. (C) PLA rolonies (white dots, Right) formed between postsynaptic cultured mouse hippocampal neurons expressing HA-NLGN + mCherry (red; Middle) and cocultured presynaptic neurons expressing myc-NRXN + GFP (green; Left). (Scale bar, 10 μm.) (D) PLA puncta in each sample (circles) and average (squares) ± SEM for indicated expression and days in vitro (color indicates data acquired on the same day). ***P < 0.001, two-way ANOVA (see Methods); *P < 0.05, one-way ANOVA (with the Tukey–Kramer post hoc test). (E) PLA reaction between endogenous GluA1 and recombinant myc in cultured rat hippocampal neurons (exposed to cLTP; see Fig. 2) expressing myc-NRXN and cytosolic fluorescent protein (green), subsequently immunostained for endogenous PSD-95 (red). (Scale bars, 10 μm, Left; 5 μm, Right.) Arrows represent dendritic spines labeled by presynaptic myc-NRXN, PSD-95, and PLA puncta. (F) PLA puncta localize to synapses. In PSD-95 and myc-NRXN channels (from E, Right), pixels were set to zero (masked) for values below a progressively increasing threshold (x axis). Puncta (PLA or randomly placed) display colocalization if nonzero pixels exist within 0.14 μm in both thresholded channels. For the indicated threshold, ∼95% of PLA but only ∼50% of randomly placed (mean ± SEM of 30 placements) puncta colocalized with pre- and postsynaptic markers. See SI Appendix, Fig. S3 and Materials and Methods for details.

Article Snippet: For SI Appendix , Fig. S2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone Labs), rabbit anti-cFos (Cell Signaling Technology, #2250); all were at 1:100 dilution.

Techniques: Binding Assay, Ligation, Amplification, Recombinant, Cell Culture, Expressing, In Vitro, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SYNPLA, a method to identify synapses displaying plasticity after learning

doi: 10.1073/pnas.1919911117

Figure Lengend Snippet: SYNPLA detects synaptic potentiation in hippocampal primary cultures and organotypic slices. (A) Diagram of SYNPLA targeting presynaptic myc-NRXN and endogenous GluA1. cLTP induces mobilization of GluA1 from an extrasynaptic pool and insertion into the postsynaptic density, decreasing the distance between the targeted proteins and permitting PLA. (B) Representative images of SYNPLA reactions performed on myc-NRXN-expressing cultured rat hippocampal neurons at 14 d in vitro for the indicated conditions; PLA is shown in gray. (Scale bar, 10 μm.) (C) Quantification of a single SYNPLA experiment (as in F). The number of SYNPLA puncta detected in each field of view (circles) and the average (squares) ± SEM across all fields of view (12 per condition) are shown. ***P < 0.001, one-way ANOVA followed by the Tukey–Kramer post hoc test. (D) Quantification of six independent experiments (indicated by different colors); the average number of SYNPLA puncta across 10 to 12 fields of view for each experiment (circles, normalized to control [CTRL]); for each experiment, a significant difference was measured between CTRL and cLTP conditions (P values between 0.00004 and 0.02), and the average PLA signal across experiments (squares) ± SEM is shown. **P < 0.01, one-way ANOVA (with the Tukey–Kramer post hoc test). (E) Diagram of SYNPLA in rat organotypic hippocampal slices. Sindbis virus expressing myc-NRXN is injected in the presynaptic CA3 region. SYNPLA between myc-NRXN and endogenous GluA1 is measured in the postsynaptic CA1 region. (F) Representative images of SYNPLA under the indicated conditions in region CA1 of organotypic slice cultures. (Scale bar, 10 μm.) (G) Quantification of SYNPLA puncta in organotypic slices from three independent experiments (indicated with different colors, four slices per condition [one circle per slice]); squares indicate average ± SEM across experiments. ***P < 0.001, paired t test.

Article Snippet: For SI Appendix , Fig. S2 different primary antibodies were used as negative controls: guinea pig anti-GluA1 (AGP-009, Alomone Labs), rabbit anti-cFos (Cell Signaling Technology, #2250); all were at 1:100 dilution.

Techniques: Expressing, Cell Culture, In Vitro, Injection

Journal: Brain

Article Title: LGI1 antibodies alter K v 1.1 and AMPA receptors changing synaptic excitability, plasticity and memory

doi: 10.1093/brain/awy253

Figure Lengend Snippet: Patient-derived LG1 IgG causes a decrease of total cell surface and synaptic AMPAR clusters in the hippocampus. (A) 3D projection and analysis of the density of total clusters of GluA1 AMPAR and PSD95, and synaptic clusters of AMPAR (defined as AMPAR clusters that co-localized with PSD95) in one of the CA3 subregions indicated in Fig. 3A, ‘Analysis’. Merged images (A, top and bottom left; GluA1 green, and PSD95 red) were post-processed and used to calculate the density of the indicated clusters (density = spots/μm3). Scale bar = 2 μm. (B–D) Quantification of the density of total (B) and synaptic (C) GluA1 AMPAR clusters, and PSD95 clusters (D) in a pooled analysis of hippocampal subregions (CA1, CA3, dentate gyrus) in animals treated with patient-derived LGI1 IgG (red) or control IgG (black) on the indicated days. Mean density of clusters in control IgG treated animals was defined as 100%. Data are presented as mean ± SEM. For each time point five animals infused with patient-derived IgG and five with control IgG were examined. Significance of treatment effect was assessed by two-way ANOVA with an alpha-error of 0.05 (asterisks) and post hoc testing with Sidak-Holm adjustment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: To determine whether the postsynaptic levels of AMPAR were affected, brain sections were incubated with a polyclonal guinea pig antibody against GluA1 (1:200, #AGP009, Alomone) for 2 h at room temperature, washed in PBS containing 0.3% Triton, and incubated with a rabbit polyclonal antibody against the postsynaptic marker PSD95 (1:250, #18258, Abcam) overnight at 4°C.

Techniques: Derivative Assay