Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, Extraction, MANN-WHITNEY, Immunostaining

Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal mouse anti-GluR2 , Neuromab , Cat# 75-002; RRID: AB_2232661.

Techniques: Plasmid Preparation, Virus, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Detailed information on the 30 primary antibodies used

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Increased sensitivity of antibodies applied on CLARITY-processed sections. a Cleared (orange) and non-cleared (blue) frontal cortex sections from case p1 were stained for CAMKIIA and imaged 15 µm deep in 1.8 µm steps using a confocal microscope. Z-level I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) are provided as single images (left). Mean intensities of each z-step were exported with LAS-X software from two stacks (cortex layer V), and the mean was plotted in dependency of the z-level (right). A side view (xz scan) is shown in the middle. Scale bars, 10 µm. b CAMKIIA-positive surfaces in the neuropil outside dendrites of cases p1 (perfusion; filled circle) and p2 (white circle) were counted in z-levels I (z = 0 µm), III (z = 3.6 µm), and VI (z = 9 µm) of two z-stacks per case and condition (acquired with identical settings). In total, two regions of interest (ROIs) were analyzed per image in Imaris with constant parameters for plus and minus CLARITY per z-plane. The mean ratios between plus and minus CLARITY (y axis) of two technical replicates are plotted; a ratio of 1 (black line) indicates identical surface numbers for plus and minus CLARITY. c Mean intensity of GLUA2 staining on case p1 was plotted in dependency of the z-level. Acquisition strategy is the same as outlined in panel (a). Scale bars, 5 µm. d More sensitive detection of GLUA2 puncta after CLARITY, analysis as described in panel (b)

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Staining, Microscopy, Software

Journal: BMC Biology

Article Title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

doi: 10.1186/s12915-023-01582-6

Figure Lengend Snippet: Super-resolution microscopy with STED and dSTORM imaging of synaptic and neuronal markers on human brain tissue after CLARITY. a Synaptic GLUA2 in the cerebral cortex acquired in confocal (upper left) and STED mode (lower right). Scale bar, 1 µm. b Insets (boxed area from (a)) provided in higher magnification unveil postsynaptic membrane morphologies like disk shape as seen in a side view on a synapse (asterisks, left column) and the detailed localization of GLUA2 within a single synaptic spot as seen in a top view on a synapse (right column). A line intensity profile (right part of (b)) across the spot surface demonstrates the intensity fluctuation along the spot (white line in the boxed areas of (b)). Mean intensities (gray values) were normalized on the highest value. a.u. = arbitrary unit. Scale bar, 1 µm. c MAP2 staining in a cerebral cortex section. STED and confocal modes of the same neuron are shown in comparison as in panel (a). Scale bar, 1 µm. d Insets (boxed area from (c)) provided in higher magnification show structural details in the STED mode. The intensity profile across the white lines in the images is plotted on the right. Scale bar, 0.5 µm. Sections for MAP2 and GLUA2 staining were derived from the superior frontal gyrus of perfusion-fixed cases. In (a)-(d), pixel size for confocal images is 50 nm. e Schematic drawing showing expected precision imaging of synaptic compartments in super-resolution images (upper left). SYP/GLUA1 co-staining in STED microscopy. The channels are shown individually (left and middle) and as overlay (right). Here, sections were incubated in hydrogel solution for two days, total clearing time was three days. Scale bars, 200 nm. f SYP/GLUA1 co-staining in dSTORM microscopy (left). An intensity profile across a synapse (rectangle in the left image, width of ten pixels) is shown on the right. Sections were incubated in hydrogel solution for one day, total clearing time was two days. Scale bar, 200 nm. All sections in (e)-(f) were derived from perfusion-fixed cases

Article Snippet: G 7 , Postsynapse (excitatory) , GLUA2 (GRIA2) , ms , 1:200 , SySy Cat# 182 111, RRID:AB_10645888 , .

Techniques: Super-Resolution Microscopy, Imaging, Membrane, Staining, Comparison, Derivative Assay, Microscopy, Incubation

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits.

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: Fig. 4 | Glioma membrane depolarization promotes glioma progression. a, Optogenetic model for glioma depolarization. Blue dots represent ChR2-expressing glioma cells; light blue rectangle denotes region of analysis. P, postnatal day. b, Proliferation index of SU-DIPG- XIII-FL-ChR2 xenografts after mock stimulation or blue light stimulation, measured as percentage of GFP+/HNA+ cells expressing Ki67 (mock stim, n = 8; stim, n = 9 mice). c, As in b but for SU-DIPG-VI-ChR2 xenografts (n = 6 mice per group). d, Representative confocal micrographs from c, illustrating proliferation of SU-DIPG-VI-ChR2 xenografts. Red denotes human nuclei staining by HNA; white denotes Ki67. Scale bar, 50 µm. e, f, Kaplan–Meier survival curves of SU-DIPG-XIII-P* (P denotes pontine tumour) xenografts that overexpress GFP-only (green) or GluA2- WT-GFP (red) (e) and GFP-only (in 80% of cells, green) or GluA2-DN- GFP (in 80% of cells, blue) (f) (n = 5 mice per group). g, Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in f, determined by

Article Snippet: We introduced SpeI (5′-TAAGCAACTAG TATGCAAAAGATTATGCAT-3′) and XmaI (5′-TGCTTACCCGGGC TAAATTTTAACACTTTCGAT-3′) restriction sites in full-length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Membrane, Expressing, Staining

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Proliferative response of GFP (control) and GluA2-dominant negative subunit expressing glioma cells (GluA2-DN) after 24-hour exposure to soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM). b , Western blot analysis of phospho-AKT (Ser473) and total AKT in GFP (control) glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm) in the presence and absence of AMPA-receptor blocker, NBQX (10μM); left. Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). c , Western blot analysis of phospho-AKT (Ser473) and total AKT in GluA2-DN expressing glioma cells in response to 5-minute exposure of soluble extracellular neuroligin-3 (NLGN3; 100nm; left). Quantitative analysis of the ratio of pAKT/AKT normalized to vehicle (right). d , Time course of evoked glioma cell EPSC block by NASPM (100 μM, duration=red bar (n=7/5 cells/mice; left); Representative trace before (black) and after (red) addition of NASPM (right). e, Quantification of (d). f , GluA2 subunit Q/R editing efficiency in SU-DIPGXIII-FL and SU-DIPGVI cells as measured by PCR and expressed as % edited. g , Expression of ADAR1, the enzyme responsible for Q/R editing of GluA2 mRNA. Plot illustrates ADAR1 enzyme mRNA expression relative to beta-actin as measured by qPCR. Analyses in a,b,c,f,g were calculated from three independent sets of cells. Data shown as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s post-hoc analysis (a,b), by two-tailed Student’s t-test (c), by two-tailed paired Student’s t-test (e). All data shown as mean ± s.e.m. *P<0.01, **P<0.001, ***P<0.001, ****P<0.0001, NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Dominant Negative Mutation, Expressing, Western Blot, Blocking Assay, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a, Electrophysiological responses by model. Number of whole cell patch clamp recordings from cells in xenografted hippocampal slices separated by electrophysiological response to local electrical stimulation. b , Demonstration of depolarizing inward current in SU-DIPXIII-FL-ChR2 cells in response to single stimulation and 20Hz pulses of blue light as measured in current clamp (top) and voltage clamp (bottom). c , Proliferation index of xenografted SU-DIPGXIII-FL-YFP control glioma cells (no opsin expressed) in response to blue light stimulation or mock stimulation as measured by the proportion of GFP+/HNA+ cells expressing Ki67 24-hours after five optogenetic stimulation sessions (n=3 mice, mock stim; n=4 mice, stim). d, Quantification of cleaved caspase-3 in xenografted SU-DIPGXIII-FL-YFP control glioma cells in response to blue light stimulation or mock stimulation as measured by total number of HNA+ cells co-labeled with cleaved caspase-3 (n=3 mice/group). e , As in (d), quantification of cleaved caspase-3 in xenografted SU-DIPXIII-FL-ChR2 glioma cells (n=3 mice, mock stim; n=4 mice, stim). f , Validation of GluA2-dominative negative AMPA receptor subunit expressing construct. Representative traces of whole-cell voltage-clamp recording of WT (black) and GluA2-DN expressing (grey) SU-DIPGVI cells in response to 500μM (S)-AMPA (n=6 cells). g , Representative traces of whole-cell voltage-clamp recording in WT (black) and GluA2-DN expressing (grey) SU-DIPGXIII-FL cells in response to 500μM (S)-AMPA (n=6 cells). SU-DIPGXIII-FL cells are unable to homogeneously express the dominant construct, and therefore may be connected to WT GluA2 expressing cells, which accounts for the remaining current in the illustrated trace. Incorporation of the GluA2-DN construct thus results in a significantly abrogated AMPAR-dependent depolarization. Data shown as mean ± s.e.m for (c,d,e). All P-values determined by two-tailed Student’s t-test. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Patch Clamp, Control, Expressing, Labeling, Biomarker Discovery, Construct, Two Tailed Test

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Optogenetic paradigm for glioma depolarization. ChR2-expressing glioma (blue), region of analysis (light blue). b , Proliferation index of SU-DIPGXIII-FL-ChR2 xenograft after mock stimulation (mock stim) or blue light stimulation (stim) measured as percent of GFP+/HNA+ cells expressing Ki67 (mock stim, n=8; stim, n=9 mice). c , As in (b), but SU-DIPGVI-ChR2 xenografts (n=6 mice/group). d , Representative confocal micrographs from (c), illustrating proliferating SU-DIPGVI-ChR2. Red=human nuclei; white=Ki67. Scale bar=50μm. e-f , Kaplan-Meier survival curves of SU-DIPGXIII-P* xenografts overexpressing e, GFP-only (green) or GluA2-WT-GFP (red) and f, GFP-only (in 80% of cells, green) or GluA2-DN-GFP (in 80% of cells, blue); n=5 mice/group. g , Competitive outgrowth of non-GluA2-DN-GFP-expressing cells in (f), determined by GFP/total human nuclei pixel intensity; (n=3 mice/group). h , Representative confocal micrographs of (f-g). White=human nuclei; green=GFP. Scale bar=50μm. i , Representative confocal images of SU-DIPGXIII-FL xenografts expressing GFP-only control (top) or GluA2-DN-GFP (bottom). Gray=MBP; White=glioma-GFP. Scale bar=500μm. j , Quantification of (i) (n=8 mice/group). k , Proliferation index of SU-DIPGVI xenografts treated with perampanel (AMPAR blocker) or vehicle control; (n=8 mice/group). l , Proliferation index of SU-DIPGXIII-FL in mice treated with meclofenamate (gap junction blocker) or vehicle control; (n=9 vehicle, n=8 treated mice). Data shown as mean±s.e.m (b,c,g,j,k,l). **P<0.01. ***P<0.001, ****P<0.0001. P-values determined by two-tailed unpaired Student’s t-test (b,c,g,k,l); two-tailed log rank analyses (e,f); two-sided Mann-Whitney test (j).

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Electrical and synaptic integration of glioma into neural circuits

doi: 10.1038/s41586-019-1563-y

Figure Lengend Snippet: a , Kaplan-Meier survival curves of second cohort of mice orthotopically xenografted with control GFP-only or GluA2-DN-GFP over-expressing cells (SU-DIPGXIII-P* xenograft model; n=5 mice per group). b , Representative coronal sections of mouse brains bearing SU-DIPGXIII-FL xenografts either expressing control GFP construct (left) or GluA2-DN-GFP construct; right). Gray, MBP; White, glioma-GFP. c , Proliferation indices of SU-DIPGXIII-FL cells at baseline in neuronal medium, in response to 10μM NBQX, in co-culture with neurons, or in co-culture with neurons in the presence of 10μM NBQX (n=3 biological replicates/group, except n=4 for baseline). d, Representative images of neuron-glioma co-cultures in the presence and absence of NBQX. Green = neurofilament (neuronal processes); Red = nestin (glioma cell processes); White = Ki67. Scale bar = 50μm. e , in vitro growth analysis of control GFP or GluA2-DN-GFP cells monitored over 3 days. f, in vitro apoptosis analysis of control GFP or GluA2-DN-GFP as measured by % of total cells co-stained with cleaved-caspase. g , 3D Matrigel invasion assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. h , Representative images of (g) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar = 1000μm. i , 3D migration assay in WT (GFP) and GluA2-DN (GluA2-DN-GFP) expressing SU-DIPGXIII-FL cells 72 hours after seeding. j , Representative images of (i) at time 0 hr (left) and 72 hr (right) in control GFP-expressing (top) and GluA2-DN-GFP expressing cells (bottom). Scale bar =1000μm. k, Representative confocal micrographs illustrating proliferating SU-DIPGVI cells in vehicle or perampanel-treated mice (n=8 mice/group). Red = human nuclei; white = Ki67. Scale bar = 50μm. l , IVIS bioluminescence analysis of overall tumor growth in SU-DIPGXIII-FL xenografts treated with vehicle or meclofenamate over a two-week period. Data represented as fold change in total flux; n=5 mice/group. Data shown as mean ± s.e.m. for (c,e,f,g,i,l). For analyses in (d-j), n=3 biological replicates. P-values determined by two-tailed log rank analyses (a), by one-way ANOVA with post-hoc analysis (c), by two-tailed unpaired Student’s t-test (f,g,i,l). *P<0.05, **P< 0.01,****P<0.0001. NS = not significant.

Article Snippet: We introduced SpeI (5 ’ -TAAGCAactagtATGCAAAAGATTATGCAT-3’) and XmaI (5 ’ -TGCTTAcccgggCTAAATTTTAACACTTTCGAT-3’) restriction sites in full length human GRIA2 clone (GluA2 WT) obtained from Origene (RC212599) by PCR.

Techniques: Control, Expressing, Construct, Co-Culture Assay, In Vitro, Staining, Invasion Assay, Migration, Two Tailed Test