polyclonal rabbit anti-human egfl7 antibody  (Reliatech)

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Immunofluorescence staining revealed EGFL7 expression in granule cells and larger blood vessels of the human dentate gyrus as well as larger neurons of the hilus. Scale bars represent 50 μm or 10 µm (magnification). ( b ) Furthermore, EGFL7 was expressed in the dentate gyrus of mice as detected by fluorescence in situ hybridization (FISH). Scale bars represent 50 µm or 10 µm (magnification). ( c ) Cells of murine hippocampi were isolated by fluorescence-activated cell sorting using a combination of the following markers: GFAP + /CD133 + /EGFR - for quiescent neural stem cells (qNSCs), GFAP + /CD133 + /EGFR + for active NSCs (aNSC), GLAST - /CD133 - /EGFR + for neural progenitor cells (NPCs), CD24 + for neuroblasts (NBs), Thy-GFP1 + for neurons and CD31 + for endothelial cells (ECs). Expression of Egfl7 was measured by quantitative reverse transcriptase-polymerase chain reaction using two housekeeping genes and data was plotted as normalized to unsorted hippocampus tissue (HC). Data are presented as mean values with 95% confidence interval (CI). ( d ) EGFL7-specific FISH probes in combination with cell type-specific markers verified EGFL7-expression in aNSCs/NPCs (Stmn1; arrowheads) and neurons (NeuN; arrowheads). Scale bars represent 10 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, Staining, Expressing, Fluorescence, In Situ Hybridization, Isolation, FACS, Reverse Transcription, Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative images of neural stem and progenitor cells isolated from mouse hippocampus (HC) and cultured as spheres. Scale bars represent 25 µm. ( b ) Size of EGFL7 -/- neurospheres was increased (58.95 ± 11.24 µm (n = 5) versus 43.85 ± 2.25 µm in wild-type control (WT); n = 4; p = 0.0317). ( c ) Fluorescence-activated cell sorting revealed an increase in activated neural stem cells (aNSCs) in EGFL7 -/- mice (5.56 ± 1.65% versus 2.20 ± 0.53% in WT, n = 3; p = 0.0499) but about equal amounts of quiescent qNSCs. ( d ) Flow cytometry-based cell cycle analysis of EGFL7 -/- and WT neurospheres yielded an increased amount of cells in the G2/M phase in EGFL7 -/- mice (33.65 ± 6.10% versus 25.10 ± 2.03%; n = 3; p = 0.0286). ( e ) The paradigm used for cell cycle analysis in vivo . ( f ) Representative images of the dentate gyrus 1 h post administration of CldU. Quantification of cells yielded an increased amount of cells in S phase in EGFL7 -/- mice (11.00 ± 2.71 versus 5.25 ± 1.71 cells per section in WT; n = 4; p = 0.0571). ( g ) Representative images of the dentate gyrus 24 h post administration of IdU and double-stained for Ki67/IdU. Quantification revealed sustained proliferation in EGFL7 -/- mice (17.25 ± 2.06 versus 10.00 ± 2.45 cells per section in WT; n = 4; p = 0.0286). Scale bars represent 90 µm. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SEM; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Isolation, Cell Culture, Control, Fluorescence, FACS, Flow Cytometry, Cell Cycle Assay, In Vivo, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Markers used in immunofluorescence analyses (IF) to label specific cell types of neural stem cell (NSC) differentiation in the dentate gyrus (DG): GFAP/Nestin for NSCs (type 1), Stmn1 for activated aNSCs and neural precursor cells (NPCs, type 1, type 2), Mash1 for aNSCs/type 2a cells, DCX for type 2b/type 3 and immature neurons, NeuN for mature neurons and granule cells. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) Quantitative analyses revealed that NSCs (GFAP + /Nestin + /BrdU + ) remained unchanged in EGFL7 -/- mice (10.50 ± 3.51 versus 11.00 ± 1.41 cells per section in wild-type controls (WT); n = 4; p > 0.9999). ( d ) The number of type 2a (BrdU + /Mash1 + ) cells was increased (11.25 ± 1.71 versus 3.00 ± 0.82 in WT; n = 4; p = 0.0286). ( e ) Type 2b/type 3 (BrdU + /DCX + ) cells were decreased (7.00 ± 2.16 versus 14.75 ± 5.56 cells per section in WT; n = 4; p = 0.0286). ( f ) The amount of aNSCs/NPCs (BrdU + /Stmn1 + ) was strongly increased in the DG of EGFL7 -/- animals (18.25 ± 4.99 versus 5.25 ± 3.59 cells per section in WT; n = 4; p = 0.0286). ( g ) Last, more newborn neurons (BrdU + /NeuN + ) were formed in the DG of EGFL7 -/- mice (21.50 ± 4.12 versus 5.75 ± 4.35 cells per section in WT; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Description of the EGFL7 fl/fl;Ascl1-CreERT2 mouse model allowing for a tamoxifen-inducible, type 2a cell-specific knock-out of EGFL7. ( b ) Experimental paradigms of applied neurogenesis assays. ( c ) The amount of aNSCs/NPCs (BrdU + /Mash1 + ) were found to be increased (5.25 ± 2.63 versus 1.00 ± 1.15 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). ( d ) The number of type 2b/type 3 cells (BrdU + /DCX + ) was decreased in EGFL7 del;Ascl1-CreERT2 mice (8.50 ± 5.51 versus 18.75 ± 2.22 in EGFL7 fl/fl ; n = 4; p = 0.0286), ( e ) while quantification of BrdU + /Stmn1 + activated neural stem and precursor cells (aNSCs/NPCs) revealed a significant upregulation in EGFL7 del ;Ascl1-CreERT2 mice (20.25 ± 3.10 versus 8.00 ± 4.24 cells per section in EGFL7 fl/fl control; n = 4; p = 0.0286). ( f ) Futhermore, the amount of adult-born neurons (BrdU + /NeuN + ) was increased (18.25 ± 6.80 versus 5.50 ± 2.65 cells per section in EGFL7 fl/fl ; n = 4; p = 0.0286). Statistical analysis was performed by Mann-Whitney U -test. Data are represented as mean ± SEM; * p < 0.05; Scale bars represent 60 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Knock-Out, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Volcano and ( b ) MA plots of RNA-sequencing-based transcriptomics in neural stem and precursor cells confirmed EGFL7 knock-out and identified an upregulation of the cytokine VEGF-D (n = 4 for each genotype; two datasets). Volcano plots illustrate the Log2 difference (i.e., fold change) versus -logP of the t-test. MA plots display the LOG2 difference versus the mean expression level (LOG2 RPKM). The VENN diagrams show the agreement of experiment 1 and 2 and the combined analysis of regulated candidates, based on P value and fold change. ( c ) Upregulation of VEGF-D upon EGFL7 knock-out was confirmed by quantitative reverse transcriptase-polymerase chain reaction (6.56 ± 2.85 versus 1.47 ± 0.61 in wild-type control (WT); n = 3; p = 0.039). ( d ) Expression of VEGF-D in the dentate gyrus of the hippocampus was visualized by immunofluorescent staining using a VEGF-D-specific antibody. Statistical analysis was performed by Student’s t-test. Data are represented as mean ± SEM; * p < 0.05. Scale bars represent 90 µm or 45 µm (magnifications).

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: RNA Sequencing, Knock-Out, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Staining

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) Representative pictures of analyzed spines of 10-week-old EGFL7 -/- and WT mice. ( b ) The analysis of DG spines of 10-week-old mice showed increased number of spines in EGFL7 -/- mice and ( c ) significantly more thin spines. ( d ) Representative pictures of analyzed spines of 20-week-old EGFL7 -/- and WT mice. ( e ) After 20 weeks the number of spines is unaffected. ( e) The numbers of thin, stubby and mushroom spines were also not affected. ( g ) The number of dendrites and their total length was found increased in EGFL7 -/- primary neuron cultures as detected by Map2 IF staining ( h ). ( i ) Timeline for analysis of neurogenesis in aged mice and quantification of BrdU + /NeuN + newborn neurons across different ages. Statistical analysis was performed by Mann–Whitney U -test. Data are represented as mean ± SD; * p < 0.05; n = 3. Scale bar represents 5 µm.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Less is more - loss of EGFL7 improves memory by upregulation of VEGF-D

doi: 10.1101/2022.04.07.487327

Figure Lengend Snippet: ( a ) In the Morris water maze (MWM), the latency to reach the visible platform, the proportion of time spent in the platform quarter and the swimming velocity were similar in both genotypes. ( b ) Body weights were similar throughout experiments. Exemplary cohort is shown as mean ± SD. ( c ) EGFL7 -/- mice reached the hidden platform faster. Escape latency was reduced considering the full-time course (AUCs) and late trials. ( d ) The proportion of time spent in the platform quarter after its removal was increased. For MWM sample sizes were n = 19 for EGFL7 -/- mice and n = 33 for wild-type littermate controls (WT). Data were compared by paired (visible platform) and unpaired two-sided Student’s t-tests, or 2-way ANOVA for time courses. ( e ) In the IntelliCage, EGFL7 -/- mice maintained longer avoidance of a previously punished corner (air puff), as revealed by a reduced proportion of nosepoke errors (n = 16 per genotype). ( f ) During preference learning in active module times (11-13:00 and 16-18:00 h each day), EGFL7 -/- mice showed faster relearning to prefer a specified corner upon switching to the opposite side (reversal learning), indicated by a higher accuracy of nosepokes (n = 16 per genotype). Overall activity is shown in ( Supplementary Fig. 8_2 ). ( g,h ) During periods in which the learning modules were inactive (doors remain closed, “default-times”), EGFL7 -/- mice retained higher preference of rewarding corners and the proportion of “memorizers” was higher, the latter defined as 35% accuracy of corner visits (random = 25%). Time course data are represented as mean ± SEM, summarizing data as mean ± SD. The boxes show the interquartile range, the line is the median, and whiskers are plotted minimum to maximum. Scatters represent individual mice. Time courses were compared by 2-way ANOVA and subsequent posthoc t-tests to assess genotype differences at specific time points without multiplicity adjustment for between group comparisons, AUCs were compared with 2-sided unpaired t-tests; * p < 0.05.

Article Snippet: As primary antibody a polyclonal rabbit anti-human EGFL7 antibody (1:50, ReliaTech GmbH) was applied.

Techniques: Activity Assay