Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the perisynaptic ECM which consists of a hyaluronan sugar matrix connected by proteoglycans including brevican, linking proteins like tenascins, and others. b) Representative images of zebrafish hindbrain at 14 and 60 days post fertilization (dpf) showing brevican (antibody staining) and hyaluronan labeled with a genetically encoded sensor ubi:ssncan-GFP . Scales: 100 µm. c) Quantification of fluorescence intensity of brevican and hyaluronan ( ubi:ssncan-GFP ) over development from 7-90 dpf. Mean fluorescence intensity for brevican and hyaluronan were normalized to the maximum intensity measured for each (90 dpf and 28 dpf, respectively). Brevican quantification, number of fish: 7 dpf, n=9; 14 dpf, n=8; 21 dpf, n=8; 28 dpf, n=7; 60 dpf, n=7; 90 dpf, n=6. For hyaluronan, number of fish: 7 dpf, n=11; 14 dpf, n=10; 21 dpf, n=6; 28 dpf, n=10; 60 dpf, n=7; 90 dpf, n=3. d) Schematic of zebrafish larval hindbrain. Gray dots indicate cell bodies and the synaptic region is shown by pink. A cholinergic neuron with a cell body and dendrites is shown by black. e) Dorsal view of zebrafish hindbrain at 10 dpf shows sparsely labeled cholinergic neurons expressing a TdTomato (TdT) tagged FingR construct that binds to the excitatory postsynaptic marker PSD-95. Tg(chata:gal4);Tg(zcUAS:PSD95.FingR-TdT-CCR5TC-KRAB(A)), hereafter abbreviated Chat-PSD95 FingR . Scale: 20 µm. f) Strategy for quantification of synapses using Chat-PSD95 FingR . Insets of region in e shows (i) several sparsely labeled neurons and (ii) a dendritic segment from one cholinergic neuron with synapses indicated by asterisks. Scales: 20 µm. g) Immunostaining for brevican protein and Chat-PSD95 FingR at 14 dpf. Synaptic region is indicated in the image. Scale: 5 µm. h) Schematic of 24 hours time lapse imaging assay to quantify changes of synapse density. i) Representative images show a single Chat-PSD95 FingR dendrite imaged at 7 dpf (t=0) and 8 dpf (t=24). Pink arrowheads: synapses present at t=0 and absent at t=24 “lost synapses”. Green arrowheads: synapses that appear at t=24 “new synapses”. White circles: present at both time points. Scale: 5 µm. j) Quantification of total excitatory synapse density and dynamics over the live imaging window of hindbrain development (5-14 dpf), based on Chat-PSD95 FingR-TdT puncta normalized to µm of dendrite length. Black line indicates static synapse density per day (p=0.3437, One-way ANOVA). Pink bars indicate lost synapses (F (4,78) p=0.0040**, One-way ANOVA). Green bars indicate new synapses (F (4,78) p=0.018*, One-way ANOVA). Asterisks in the figure represent results of Tukey’s multiple comparisons with respect to the 5-6 dpf. Number of fish: 5-6 dpf, n=15; 7-8 dpf, n=18; 9-10 dpf, n=18; 11-12 dpf, n=15; 13-14 dpf, n=17. Results from individual fish in . k) Schematic of time lapse imaging to determine the fate of individual synapses at the indicated timepoints. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments were performed at 10-12 dpf. l) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6, which disappeared between t=12 and t=24. Left shows a low power image of a Chat-PSD95 FingR cholinergic neuron. Dashed square indicates inset. Inset shows raw fluorescence (top) and fluorescence overlaid with 3D reconstruction of synapses (bottom). Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 5 µm in low power image and 2 µm in inset. m) Kaplan-Meier plot of survival of individual synapses over time (Data from n=19 fish, n=427 stable synapses and n=25 new synapses). n) Quantification of the distance from the nearest synapse for stable and new synapses at t=6 (Stable, n=116 inter-synapse intervals from n=14 fish; New, n=25 inter-synapse intervals from n=14 fish; p<0.0001****, Welch’s t-test, performed for synapses). The inter-synapse intervals were normalized by the mean of stable synapses for each cell. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Staining, Labeling, Fluorescence, Expressing, Construct, Marker, Immunostaining, Imaging

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of brevican and hyaluronan ( ubi:ssncan-GFP ) in the hindbrain at 7, 14, 28, and 60 dpf. Scales: 100 µm. b) Representative image of PNN (perineuronal net)-like brevican staining at 60 dpf. Square indicates the region of inset on the right. Arrowheads indicate PNN-like brevican signals. Scale: 100 µm.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Staining

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of strategy for ECM digestion by hyaluronidase injection into the hindbrain ventricle. Tissues fixed or imaged 6 hours after injection with hyaluronidase or vehicle (PBS). b) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT after vehicle or hyaluronidase injection. Scale: 5 µm. c) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish (vehicle, n=14 fish; hyaluronidase, n=17 fish; p=0.020*, Welch’s t-test). d) Representative merged images of Chat-PSD95 FingR collected before injection (t=0, pink) and 6 hours after (t=6, green) hyaluronidase or vehicle injection. Overlap of pink and green appears white. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. e) Quantification of newly observed, lost, or stable synapses between t=0 and t=6 after hyaluronidase vs. vehicle injection (vehicle, n=17 fish; Hyal, n=16 fish; p=0.017* for newly observed, p=0.117 for lost, p=0.722 for stable, Welch’s t-test). f) Schematic of generation of brevican knock-out ( bcan -/- ) fish by CRISPR genome editing. Guide RNAs targeting exon 3 and exon 14 were injected to delete 19 kbp of the bcan gene. The truncation resulted in loss of bcan mRNA and brevican protein (see Extended Data Fig 3e,f). g) Representative images of Chat-PSD95 FingR dendrites at 14 dpf from fixed section stained for ΤdT from bcan +/+ vs. bcan -/- fish. Scale: 5 µm. h) Quantification of synapse density at 14 dpf (Chat-PSD95 FingR puncta) per µm of dendrite length. Dots represent means per fish, with at least 1 dendrite quantified per fish ( bcan +/+ , n=16 fish; bcan -/- , n=14 fish; p=0.031*, Welch’s t-test). i) Representative merged images of Chat-PSD95 FingR collected from bcan +/+ and bcan -/- at t=0 (pink) and t=24 (green). Pink arrowheads: lost synapses, green arrowheads: new synapses. Experiment performed at 10-12 dpf. Non-merged images in . Scale: 5 µm. j) Quantification of newly observed, lost and stable synapses between t=0 and t=24 bcan +/+ vs. bcan -/- fish ( bcan +/+ , n=13 fish; bcan -/- , n=15 fish; p=0.010* for newly observed, p=0.288 for lost, p=0.085 for stable, Welch’s t-test). Values were plotted as mean ±SEM. *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Injection, Staining, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images and quantification of hyaluronan depletion after hyaluronidase injection at 14 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) at 6 hours post injection (hpi) (vehicle, n=7 fish; Hyal, n=8 fish; p=0.0023**, Welch’s t-test) and 24 hpi (vehicle, n=5 fish; Hyal, n=4 fish; p=0.392, Welch’s t-test). GFP intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 100 µm. b) Representative images and quantification of hyaluronan depletion at 6 hpi at 60 dpf, measured as mean fluorescence intensity of GFP ( ubi:ssncan-GFP ) nomalized to vehicle control (vehicle, n=3 fish; Hyal, n=3 fish; p=0.049*, Welch’s t-test). Scale: 100 µm. c) Representative images and quantification of brevican depletion at 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=19 fish; Hyal, n=18 fish; p=0.0025**, Welch’s t-test). Brevican intensity normalized to mean of vehicle control. Dashed lines indicate hindbrain regions. Scale: 20 µm. d) Representative images and quantification of SV2 presynapse marker 6 hours after hyaluronidase injection at 14 dpf (vehicle, n=7 fish; Hyal, n=8 fish; p=0.003**, Welch’s t-test). SV2 intensity normalized to mean of vehicle control. Scale: 100 µm. e) Non-merged images of time lapse imaging from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. f) Expression of bcan gene in bcan -/- fish assessed by RT-qPCR from whole larvae. Expression normalized to ef1a housekeeper gene (n=3/group; p=0.0064**, Welch’s t-test). g) Representative images and quantification of brevican protein in bcan -/- fish ( bcan +/+ , n=10 fish; bcan -/- , n=12 fish; p<0.0001****, Welch’s t-test). Brevican intensity normalized to mean of bcan +/+ control. Scale: 100 µm. h) Representative images and quantification of SV2 presynapse marker in bcan -/- fish at 14 dpf ( bcan +/+ , n=15 fish; bcan -/- , n=15 fish, p=0.049*, Welch’s t-test). SV2 intensity normalized to mean of bcan +/+ control. Scale: 100 µm. i) Non-merged images of time lapse imaging experiment from . Circles indicate stable synapses. Boxes indicate the region shown in . Scale: 5 µm. Values were plotted as mean ±SEM. ****: p<0.0001; **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Injection, Fluorescence, Control, Marker, Imaging, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of microglial ablation by adding metronidazole (Mtz) to fish water in fish that express nitroreductase ( ntr ) in microglia and macrophages ( Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) ). b) Representative images of brevican in synaptic region after vehicle (DMSO) or microglial ablation with 5mM metronidazole (Mtz) in fish water for 24 hours. Experiment at 14 dpf. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of vehicle control (vehicle, n=15 fish; Mtz, n=11 fish; p=0.043*, Welch’s t-test). d) Heatmap of absolute expression (counts) of mmp , adam , and adamts metalloproteinase genes in zebrafish microglia from bulk RNAsequencing at 28 dpf in optic tectum (OT), midbrain (MB) and hindbrain (HB). Marker genes hexb and p2ry12 shown for comparison. Reanalyzed from . e) Schematic of a dimer of the membrane-associated metalloproteinase MMP14 and design of the mmp14b-HA expression construct for epitope tagging of the C-terminal end with HA. Catalytic domain, transmembrane domain and cytoplasmic domain are indicated. f) Mmp14b-HA transgene expression in a mpeg-GFP microglia expressing mpeg:mmp14b-HA. Inset shows HA signals colocalized with microglial processes (arrow heads). Scale: 5 µm. g) Quantification of Mmp14b-HA intensity in microglial processes vs soma. Lines connect data from the same microglia (n= 6 microglia from 3 fish; p=0.015*, Paired t-test per cell). h) Representative images of brevican staining in synaptic regions of mmp14b -/- and mmp14b +/+ control at 14 dpf. Scale: 20 µm. i) Quantification of brevican intensity in synaptic region normalized to mean of mmp14b +/+ control ( mmp14b +/+ , n=10 fish; mmp14b -/- , n=11 fish; p=0.0014**, Welch’s t-test). j) Representative images of microglia-specific Mmp14b rescue experiment. Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14 -/- fish were injected with mpeg:mmp14b-HA construct at one-cell stage embryos and analyzed at 14 dpf. Scale: 5 µm. k) Quantification of synapse density (Chat-PSD95 FingR puncta per µm of dendrite length) in mmp14b +/+ and mmp14 -/- with or without microglial-specific rescue with mpeg:mmp14b-HA. Dots show means per fish from at least 1 dendritic segment analyzed per fish ( mmp14 +/+ no rescue, n=16 fish; mmp14b +/+ rescue, n=14 fish; mmp14b -/- no rescue, n=16 fish; mmp14b -/- rescue, n=18 fish; Two-way ANOVA, F (1,60) interaction effect p=0.0022**, asterisks show Tukey’s multiple comparisons). l) Schematic of time lapse imaging to measure synapse turnover in Chat-PSD95 FingR ;mmp14b +/+ and ;mmp14b -/- fish. m) Representative merged images of synapses in Chat-PSD95 FingR ;mmp14b -/- and ; mmp14b +/+ fish at t=0 (pink) and t=24 (green). Experiments at 10-12 dpf. Pink arrowheads: lost synapses, green arrowheads: newly observed synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, and stable synapses between t=0 and t=24 in mmp14b +/+ vs mmp14b -/- fish ( mmp14b +/+ , n=14 fish; mmp14b -/- , n=16 fish; p<0.0001**** for newly observed, p=0.058 for lost, 0.61 for stable, Welch’s t-test). o) Schematic of time lapse imaging to determine the fate of individual synapses in mmp14b -/- fish. Synapses at t=0 were defined as “stable” synapses. Synapses born between t=0 and t=6 were defined as “new” synapses and subsequently followed with the 6 hour timepoint set as t=0 (lower timecourse). Experiments performed at 10-12 dpf. p) Representative image of a single excitatory synapse imaged at t=0, 6, 12, and 24 shows a “new synapse” born between t=0 and t=6 in mmp14b -/- fish. Raw fluorescence images on top and fluorescence overlaid with 3D reconstruction of synapse on bottom. Arrowheads: newborn synapse. Circle: site of newborn synapse. Scale: 2 µm. q) Kaplan-Meier plot of survival of individual synapses over time in mmp14b +/+ control (from ) and mmp14b -/- fish (For mmp14b -/- , Data from n=15 fish, n=331 stable synapses and n=26 new synapses). Values were plotted as mean ±SEM. ****: p<0.0001; **: <0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Control, Expressing, Marker, Comparison, Membrane, Construct, Staining, Injection, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Representative images of mCherry and the microglial marker 4C4 and quantification of microglia number in the Tg(mpeg:gal4);Tg(UAS:NTR-mCherry) with Mtz or vehicle (DMSO) treatment at 14 dpf (vehicle, n=3 fish; Mtz, n=3 fish; p=0.015*, Welch’s t-test). non-Tg= non-transgenic fish. Scale: 100 µm. b) Representative images and quantification of brevican staining in Mtz-treated non-transgenic siblings at 14 dpf (vehicle, n=6 fish; Mtz, n=6 fish; p=0.412, Welch’s t-test). Brevican intensity normalized to the mean of vehicle control. Scale: 20 µm. Values were plotted as mean ±SEM.*: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Marker, Transgenic Assay, Staining, Control

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Scatter plot of gene expression (sorted by rank) of iPSC derived microglia and human fetal microglia. MMPs, ADAMs, ADAMTSs families that have transmembrane domain in dark green; MMPs, ADAMs, ADAMTSs without transmembrane domains in light green; microglial marker genes in violet. Data reanalyzed from the previous study . b) Schematic of the iPSC derived astrocyte-neuron-microglia tri-culture system. c) Schematic of the proteomics analysis with iPSC derived cells. Cell supernatants were collected from co-culture (neurons and astrocytes; no microglia) and tri-culture (neurons, astrocytes and microglia; with microglia) and analyzed. d) Venn diagram of proteins detected in cell culture supernatants with or without microglia. Proteins only detected in the absence of microglia are listed on the left and proteins only detected in the presence of microglia are listed on the right. ECM related proteins as defined by the matrisome database MatrisomeDB2.0 are labeled in red. e) Volcano plot of proteins in the intersection of venn diagram in d. The fold change is calculated by comparing the “with microglia” condition to the “no microglia” condition. Thresholds: p-value<0.05 (horizontal dashed line), and the vertical grey dash lines show where the average log 2 fold change >1 (vertical dashed lines). ECM related proteins as defined by the matrisome database Matrisome DB2.0 in red. Significantly matrisome proteins are outlined in blue. f) Strategy for MMP14 knockdown (KD) by shRNA (short hairpin RNA interference) with a scrambled shRNA control, and MMP14 rescue by expression of MMP14 fused to GFP, vs a GFP only control in microglia (MG). The rescue construct has silent mutations in the MMP-GFP to prevent knockdown by shMMP14. All constructs were delivered by lentivirus at the iPSC stage and used the ubiquitous promoter EF1a. g) Representative images of the tri-culture system with MMP14 KD and rescue in microglia. Iba1 (microglia), S100β (astrocyte), and neurons (MAP2) stainings are shown. Scale: 100 µm. h) Quantification of the process length of microglia in the tri-culture system with MMP14 KD and rescue. Dots show mean microglial process length per field of view from 18 fields of view over 3 independent experiments. Box-and-whiskers plot: box shows 25-75th percentile, whiskers show min to max. Statistics performed on means per field of view. (Kruskal-Wallis test, asterisks in the figure show results of Dunn’s multiple comparisons). i) Representative image of western blotting for brevican in cell supernatant and MAP2 loading control in cell lysate without microglia or with microglia after MMP14 KD and rescue. j) Quantification of brevican in the cell supernatant without microglia or with microglia after MMP14 KD and rescue. Each dot represents mean brevican intensity normalized to no microglia control from n=4 independent experiments (One-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparisons). Values were plotted as mean ±SEM. ****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Gene Expression, Derivative Assay, Marker, Co-Culture Assay, Cell Culture, Labeling, Knockdown, shRNA, Control, Expressing, Construct, Western Blot

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) MMP14 mRNA levels from MMP14 KD microglia analyzed by RT-qPCR showing two independent shRNAs targeting MMP14, with scrambled shRNA as control. Expression normalized to GAPDH housekeeping gene (n=3 independent experiments; One-way ANOVA with Tukey’s multiple comparison). b) MMP14 mRNA levels from MMP14 KD and MMP14-GFP rescue microglia analyzed by RT-qPCR. Expression normalized to GAPDH (n=3 independent experiments; one-way ANOVA, asterisks in the figure show results of Tukey’s multiple comparison). c) Western blot of MMP14 showing MMP14 KD and rescue with MMP14-GFP in microglia. Note higher molecular weight of MMP14-GFP vs. endogenous MMP14. d) Quantification of the microglia cell number in the triculture system with MMP14 KD and MMP14-GFP rescue (n=3 independent experiments, p=0.209, one-way ANOVA). e) Representative western blot and quantification of Brevican in cell supernatant vs. MAP2 loading control in cell lysate from the triculture system with MMP14 KD (n=4 independent experiments, One-way ANOVA, asterisks show results of Tukey’s multiple comparison). Values were plotted as mean ±SEM.****: p<0.0001; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Quantitative RT-PCR, shRNA, Control, Expressing, Comparison, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Extracellular matrix proteolysis maintains synapse plasticity during brain development

doi: 10.1101/2025.02.27.640672

Figure Lengend Snippet: a) Schematic of the forced swim paradigm performed in this study. Forced swim (FS) groups were put on a petri dish with a stirrer bar and exposed to water current for 8 hours per day at 650 rpm, for two consecutive days. Control (CT) groups were put in a petri dish without stirrer bars. Experiments were performed at 13-14 dpf. b) Representative images of brevican staining in control and forced swim groups. Scale: 20 µm. c) Quantification of brevican intensity in synaptic region normalized to mean of control group (CT, n=16 fish; FS, n=17 fish; p=0.0021**, Welch’s t-test). d) Representative images of Chat-PSD95 FingR dendrites from fixed sections stained for TdT in forced swim or control group. Scale: 5 µm. e) Quantification of excitatory synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length in forced swim or control group. Dots represent means per fish from at least one dendritic segment analyzed per fish (CT, n=21 fish; FS, n=21 fish; p=0.026*, Welch’s t-test). f) Schematic of time lapse synapse imaging performed before (t=0) and after (t=30) forced swim paradigm. Experiments were performed at 10-12 dpf. g) Representative merged images of time lapse assay in the Chat-PSD95 FingR before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merge images in Extended Data Fig.10a. Scale: 5 µm. h) Quantification of newly observed, lost, and stable synapses after forced swim paradigm (CT, n=8 fish; FS, n=9 fish; p=0.0060** for newly observed, p=0.140 for lost, p=0.507 for stable, Welch’s t-test). i) Representative images of brevican staining in mmp14b -/- fish from forced swim or control group. Scale: 20 µm. j) Quantification of brevican intensity in synaptic region in mmp14b -/- fish normalized to control group (CT, n=13 fish; FS, n=11 fish; p=0.397, Welch’s t-test). k) Representative images of Chat-PSD95 FingR dendrite in mmp14b -/- fish from forced swim or control group. Scale: 5 µm. l) Quantification of synapse density (Chat-PSD95 FingR puncta) per µm of dendrite length from forced swim or control group in mmp14b -/- . Dots represent means per fish from at least 1 dendritic segment analyzed per fish (CT, n=13 fish; FS, n=16 fish; p=0.48, Welch’s t-test). m) Representative merged images of time lapse assay in the Chat-PSD95 FingR ;mmp14b -/- fish before (t=0, pink) and after (t=30, green) forced swim paradigm or control. Pink arrowheads: lost synapses, green arrowheads: new synapses. Non-merged images in . Scale: 5 µm. n) Quantification of newly observed, lost, stable synapses after forced swim in the mmp14b -/- fish (CT, n=14 fish; FS, n=13 fish; p=0.542 for newly observed, p=0.621 for lost, p=0.690 for stable, Welch’s t-test). Values were plotted as mean ±SEM. **: p<0.01; *: p<0.05; ns: not significant.

Article Snippet: To obtain anti-brevican antibody, SI10-brevican (Addgene, #46300) was transfected into HEK293 cells by Lipofectamine 3000 Transfection reagent (Thermo Scientific) and collected culture media at 48 or 72 hours after transfection.

Techniques: Control, Staining, Imaging

Journal: PLoS ONE

Article Title: Combined proteomic and metabolomic analyses of cerebrospinal fluid from mice with ischemic stroke reveals the effects of a Buyang Huanwu decoction in neurodegenerative disease

doi: 10.1371/journal.pone.0209184

Figure Lengend Snippet: Characterization of selected protein candidates in CIR–induced, sham and BHD treatment ischemic stroke mice.

Article Snippet: The following primary antibodies were used: anti-amyloid beta precursor like protein (APLP1), anti-stathmin 1 (STMN1), anti-dickkopf-related protein 3 (DKK3), anti-carboxylesterase 1C (CES1C), anti-serpin family B member 5 (SERPINB5), and anti-alpha-2-macroglobulin (A2M) (GeneTex); anti-secretogranin 3 (SCG3), anti-brevican (BCAN), and anti-neuronal pentraxin receptor (NPTXR) (Santa Cruz Biotechnology); anti-cell adhesion molecule 4 (CADM4) and anti-epiplakin 1 (EPDR1) (Abcam); and anti-heat shock protein family A (Hsp70) member 1A (HSPA1A), anti-junction plakoglobin (JUP), and anti-S100 calcium binding protein B (S100B) (Abnova).

Techniques: Coagulation, Glycoproteomics, Ubiquitin Proteomics

Journal: Nature

Article Title: Required growth facilitators propel axon regeneration across complete spinal cord injury

doi: 10.1038/s41586-018-0467-6

Figure Lengend Snippet: a, Laminin immunohistochemistry (IHC) images plus quantification (mean±SEM stained area), and dot blot plus quantification (mean±SEM density). TP, total protein. (* P <0.01, one-way ANOVA/Bonferroni). b, IHC images and quantification (mean±SEM cell number) of astrocyte proliferation and density. (ns non-significant, * P <0.0005, one-way ANOVA/Bonferroni). c, BDA-labeled axon regrowth past PB and in LC among CD13-positive stromal cells (left) and along laminin (right). White arrows denote PB. d-f, IHC images (d) and graphs of mean±SEM axon contact with laminin (e) (*** P <0.0001; Student’s two-tailed t-test, t (9)=107.4), and mean±SEM axon length per tissue volume (f) (* P <0.0005 one-way ANOVA/Bonferroni). g, CSPG dot blot (mean±SEM density). (ns non-significant, * P <0.05, one-way ANOVA/Bonferroni). For all graphs, dots show n mice per group. h, i, BDA-labeled axon regrowth through astrocytes of PB (h) and along laminin in LC (i) in spite of dense brevican (BCAN).

Article Snippet: Primary antibodies were: rabbit anti-GFAP (1:2000; Dako, Santa Clara, CA); rat anti-GFAP (1:1000, Thermofisher, Grand Island, NY); chicken anti-GFAP (1:1000, Novus Biologicals, Littleton, CO); rabbit anti NeuN (1:1000, Abcam, Cambridge, MA); rabbit anti-GDNFR-a (GDNF-receptor alpha) (1:1000, Abcam, Cambridge, MA); sheep anti-BrdU (1:300, Maine Biotechnology Services, Portland, ME); rabbit anti-HSV-TK (1:1,000, , ); goat anti-CD13 (1:1000, R&Dsystems, Minneapolis, MN); rabbit anti-Laminin 1 (1:100, Sigma, St.Louis, MO); rabbit anti-Fibronectin (1:500, Millipore, Burlington, MA); rabbit anti-Collagen 1a1 (1:300, Novus Biologicals, Littleton, CO); mouse anti-NeuN (1:2000, Millipore, Burlington, MA); mouse anti-CSPG (1:100, Sigma); rabbit anti-Brevican (BCAN) (1:300, Novus Biologicals, Littleton, CO); guinea pig anti-NG2 (CSPG4) (Drs. E.G. Hughes and D.W. Bergles , Baltimore, MA); rat anti-PECAM-1 (1:200, BD Biosciences, San Jose, CA); guinea pig anti-homer1 (1:600, Synaptic Systems GmbH, Germany); rabbit anti-Synaptophysin (1:600,Dako, Santa Clara, CA); rabbit anti-RFP (1:1000, Rockland,Limerick,PA); chicken anti-RFP (1:500, Novus Biologicals, Littleton, CO); goat anti-GFP (1:1000, Novus Biologicals, Littleton, CO).

Techniques: Immunohistochemistry, Staining, Dot Blot, Labeling, Two Tailed Test