bsa  (Alomone Labs)

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: In the 8-days-old wild type mice (P8), SorCS2 staining colocalized with astrocytic marker, glial fibrillary acidic protein (GFAP), and surrounded vascular endothelial cells labeled with α-d-galactosyl-specific isolectin B4 (IB4), as indicated (A). Sorcs2 -/- abolished SorCS2 specific staining in the brain of P8 mice, while GFAP and IB4 staining was not affected (bars correspond to 20 μm). An intensive aquaporin-4 (AQP4) and SorCS2 staining suggests astrocytic endfeet surrounding IB4 positive vascular endothelial cells in the brain of P8 wild type mice (B; bars correspond to 50 μm). Merged image suggests colocalization of AQP4 and SorCS2 in astrocytic endfeet surrounding blood vessel, as indicated by arrows in the magnified image (bar is 10 μm).

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Staining, Marker, Labeling

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: Brain slices were stained with glial fibrillary acidic protein (GFAP) to stain astrocytes, SorCS2 and vascular endothelial cell label α-d-galactosyl-specific isolectin B4 (IB4), as indicated. In 2-month-old wild type mice, some GFAP staining was co-localized with SorCS2 staining as indicated by arrows. No SorCS2 staining was seen in brain slices from 2-month-old Sorcs2 -/- mice. Bars correspond to 50 μm.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Staining

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: An averaged blood flow map (gray scale) with an overlaid relative cerebral blood flow response (rCBF; color) to whisker stimulation, which induced a local change in blood perfusion of the primary somatosensory cortex in wild type (A) and Sorcs2 -/- mice (B). Bars correspond to 1000 μm. Increase in parenchymal perfusion of somatosensory cortex in response to contralateral whisker stimulation was reduced in Sorcs2 -/- mice ( n = 9) compared to wild type mice ( n = 8; C). Responses in (C) were compared using two-way ANOVA. *, P < 0.05.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Whisker Assay

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: Laser Speckle contrast imaging was used to assess changes in arterial diameter and arterial blood flow increase in response to whisker stimulation in wild type (WT) (A) and Sorcs2 -/- mice (B). Color scale shows blood flow index (BFI). Bar correspond to 1000 μm. The red rectangle identifies the 3 rd branch of middle cerebral artery used for single vessel analysis. Representative single artery segmentation profiles show changes in diameter and blood flow in response to whisker stimulation, as indicated in X-axis (C). Color code corresponds to BFI. Y-axis indicates cross-section coordinate in respect to center of the vessel, i.e., inner diameter can be identified. There was no difference in baseline diameter of the 3 rd branch of middle cerebral artery between Sorcs2 -/- and WT mice (D). Averaged peak responses to whisker stimulation revealed that Sorcs2 -/- mice showed smaller increase in diameter (E) and blood flow (F) in the 3 rd branch of middle cerebral artery compared to WT. * and **, P < 0.05 and 0.01 (unpaired t -test). n = 7 - 9.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Imaging, Whisker Assay

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: Brain slices were loaded with Ca 2+ sensitive dye, Calcium Green-1/AM and that was preferentially allocated to astrocytes. White arrows indicate astrocytic endfeet surrounding a parenchymal arteriole (A). Electric field 2+ stimulation increased intracellular Ca 2+ in astrocytes and this increase was larger in the brains from wild type mice ( n = 5) than in brain slices from Sorcs2 -/- mice ( n = 4) (B). ** P < 0.01 (two-way ANOVA with Sidak’s multiple comparisons test).

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques:

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: The diameter of isolated middle cerebral arteries from wild type (WT) and Sorcs2 -/- mice was not different (A). There was no difference in the vasorelaxation in response to 10 -5 M carbachol between wild type and Sorcs2 -/- arteries. n = 8 – 11. P < 0.001.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Isolation

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: This plot depicts fold changes in protein expression in astrocytes from Sorcs2 -/- vs. wild type mice correlated to the probability that the protein is differentially expressed. The comparisons were done for astrocytes isolated from P8 mice (A) and 2-month-old mice (B). P < 0.05 was set as the significant threshold for differential expression and depicted on the graphs with horizontal dotted line. Dots in red denote significantly upregulated proteins, dots in blue denote significantly downregulated proteins. Gray dots indicate proteins that were not significantly different. Venn diagram (C) shows the number of overlapping differently expressed proteins, which expression was significantly changed in P8 and 2-month-old Sorcs2 -/- mice vs. wild type mice. n = 4.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Expressing, Isolation

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet:

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Expressing, Comparison, Control

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: Several differentially expressed proteins in astrocytes from P8 (A) and adult (B) mice are involved in glutamatergic signaling. These proteins were either downregulated (blue) or upregulated (red) or unchanged (grey) in Sorcs2 -/- mice in comparison with wild type. See also Supplementary excel files 1 and 2. EAAT1, excitatory amino acid transporter 1; ASCT1, alanine serine cysteine transporter 1; mGluR3, metabotropic glutamate receptor 3; GluR2, glutamate ionotropic receptor AMPA type subunit 2; NMDAR1, N-methyl-D-aspartate receptor (NMDAR) subunit 1; Kir6.2, ATP-sensitive K + channel pore subunit 6.2; CaV2.1, P/Q voltage-dependent calcium channel; CaMKII, Calcium/calmodulin-dependent protein kinase II; CaM, calmodulin; SERCA2, sarco/endoplasmic reticulum Ca 2+ -ATPase 2; MICU3, mitochondrial calcium uptake protein 3; Stx1, syntaxin-1; Syt1, synaptotagmin 1; Synj1, synaptojanin 1; Got2, glutamic-oxaloacetic transaminase 2; and Glud1, glutamate dehydrogenase 1.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: Comparison

Journal: bioRxiv

Article Title: SorCS2 modulates neurovascular coupling via glutamatergic and calcium signaling in astrocytes

doi: 10.1101/2023.02.16.528727

Figure Lengend Snippet: Gene ontology pathways were analyzed for enrichment of differentially expressed proteins. A negative z-score (blue) indicates suppression of the pathway and a positive z-score (red) indicates enhancement in Sorcs2 -/- mice. The significance of changes is predicted by the Ingenuity Pathway Analysis (Qiagen). See also Expanded View Table 3. The horizontal dashed line indicates P = 0.05.

Article Snippet: The brain slices were incubated for 30 min in 1% bovine serum albumin (BSA) in PBS with 0.1% Triton X-100 (Tx), followed by overnight incubation at 4°C in 50 nM Trisbased buffer solution with 1% BSA containing anti-SorCS2 antibody (dilution 1:100; AF4237, R&D, Minneapolis, MN, USA), anti-Aquaporin4 antibody (1:2000; AQP-014, Alomone, Jerusalem, Isreal), Alexa Fluor® 647 galactosyl-specific isolectin (1:200; I32450, Thermo Fischer, Waltham, MA, USA), and anti-GFAP antibody (1:1000; Z0334, Dako).

Techniques: