Journal: bioRxiv

Article Title: Reduced Folate Carrier 1 (RFC1/Slc19a1) Suppression Exacerbates Blood-Brain Barrier Breakdown in Experimental Ischemic Stroke in Adult Mice

doi: 10.1101/2024.10.28.620539

Figure Lengend Snippet: A) PFA fixated 20 µm-thick brain sections of naive Swiss Albino mice was labelled with anti-RFC1 antibody (red), and α-SMA (green). RFC1 is expressed in α-SMA positive precapillary arterioles (arrows) as well as capillaries (arrowheads). B) RFC1 is not expressed in AQP4 positive astrocytic perivascular endfeet rather the RFC1 immunoreactivity is observed in spatially distinct regions. C) Isolated brain microvessel (< 9 μm diameter) preparations were immunohistochemically labelled with anti-RFC1 antibody (red), and colocalized with the vessel marker Lectin (green). The bump-on-a log pericyte shape can be observed as RFC1 positive (yellow arrows). D) The common pericyte marker PDGFR-β (green) colocalized with RFC1. Yellow arrow depicts a pericyte body appearing as a bump-on-a log on the microvessel. E) CD13 (green) also colocalized with RFC1. Nuclei were labeled with Hoechst 33258 (blue). Scale bars: 10 μm. F) RFC1 labeling in the post-mortem human cortex also showed that RFC1 is expressed in the endothelial cells and pericytes (yellow arrow) ( www.proteinatlas.org ). Scale bars: 20 μm

Article Snippet: Next, they were incubated overnight at + 4°C with primary antibodies against RFC1 (SLC19A1, MyBioSource MBS9134642 and Sigma-Aldrich AV44167; both produced in rabbit); for mural cells against PDGFR-β (R&D Systems, AF1042), CD13 (Acris Antibodies, AM26636AF-N); for endothelium CD31 (BD Bioscience, 550274).

Techniques: Isolation, Marker, Labeling

Journal: Acta neuropathologica

Article Title: Blood-spinal cord barrier breakdown and pericyte reductions in amyotrophic lateral sclerosis.

doi: 10.1007/s00401-012-1039-8

Figure Lengend Snippet: Fig. 4 Pericyte capillary reduction in ALS spinal cord correlates with magnitude of vascular disruption. a Representative confocal micros- copy analysis of PDGFRb-positive pericytes (green) and lectin- positive capillaries (red) in NNDC and sporadic ALS cervical spinal cord anterior horn. b Quantification of PDGFRb-positive pericyte capillary coverage in cervical spinal cord anterior horn. Mean ± SEM, n = 5 NNDC, eight sporadic (sALS) and three familial (fALS)

Article Snippet: Sections were then incubated overnight at 4 C in the following primary antibodies: goat anti-platelet derived growth factor b (PDGFRb) (10 lg/mL, R&D systems), goat anti-hemoglobin (10 lg/ml, R&D systems), mouse anti-glycophorin A (CD235a) (1:100, Dako, Carpinteria, CA, USA), rabbit anti-fibrinogen (1:100, Dako), goat anti-thrombin (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-neuronal specific antigen A60 (NeuN) (EMD Millipore, Billerica, MA, USA).

Techniques: Disruption

Journal: The FASEB Journal

Article Title: Pericyte dynamics in the mouse germinal matrix angiogenesis

doi: 10.1096/fj.202200120r

Figure Lengend Snippet: FIGURE 3 PDGFRβ-positive pericyte coverage is similar between the mouse GM and the developing cerebral CTX. (A,B) High magnification images of the developing cortex (CTX) and the germinal matrix/postnatal subventricular zone (GM/SVZ) stained for blood vessels using Isolectin B4 (IB4, blue), and for pericytes (anti-PDGFRβ, red). Purple dashed lines mark the CTX, and green dashed lines highlight the GM/SVZ regions. Yellow asterisk indicates IB4 labeled macrophages at E10.5 in the CTX. Cropped individual images (white solid boxes) of the endothelium and PDGFRβ-positive pericytes are displayed below each timepoint to best visualize pericyte coverage of the blood vessels (A′,B′). (C) Example of data and method used to quantify pericyte coverage (see Materials and Methods). (D) Quantification of pericyte coverage reveals no significant differences between the CTX and the GM. Scatter dot plot shows mean values with standard deviation n = 5 per time point except E10.5 (n = 4) and P5 (n = 3). Data was analyzed using a two-way anova with Bonferroni's multiple comparison test. LV, lateral ventricles; E, embryonic; P, postnatal. Scale bar, 50 µm

Article Snippet: Samples were then immunostained in PBLEC (5% Triton X- 100, 1 M MgCl2, 1 M CaCl2, and 1 M MnCl2 in 1X PBS) overnight at 4°C with Biotinylated IB4 (1:50; Vector Laboratories; Cat# BAF1042) and/or the following antibodies: goat anti- PDGFRβ (1:100; R&D Systems; Cat# BAF1042), rabbit anti- NG2 (1:700; Millipore; Cat# AB5320), rabbit anti- Desmin (1:500; Abcam; Cat# AB15200), anti– αSMA- FITC (1:200; Millipore Sigma, Cat# F3777).

Techniques: Staining, Labeling, Standard Deviation, Comparison

Journal: The FASEB Journal

Article Title: Pericyte dynamics in the mouse germinal matrix angiogenesis

doi: 10.1096/fj.202200120r

Figure Lengend Snippet: FIGURE 4 Delayed NG2 expression in pericytes of the mouse GM compared to the developing cerebral CTX. (A,B) High magnification images of the developing cortex (CTX) and the germinal matrix/postnatal subventricular zone (GM/SVZ) stained for blood vessels using Isolectin B4 (IB4, blue) and for pericytes with anti-PDGFRβ (red) and anti-NG2.63 Purple dashed lines mark the CTX, and green dashed lines highlight the GM/SVZ regions. Cropped individual images (white solid boxes) of the endothelium and pericytes are displayed below each timepoint to best visualize the overlap of NG2 and PDGFRβ expression and pericyte coverage of the blood vessels (A′,B′). Note that in the CTX and the GM/SVZ, NG2 is also expressed by oligodendrocyte precursor cells beginning at E14.5; these cells were excluded from the analysis. (C) Quantification of NG2-positive pericyte coverage reveals reduced coverage at E11.5 in the GM. Scatter dot plot shows mean values with standard deviation. n = 5 per time point except P5 (n = 3). Data was analyzed using a two-way anova with Bonferroni's multiple comparison test. *p < .05. (D) Quantification of NG2+; PDGFRβ− (gray), PDGFRβ+; NG2− (red) and PDGFRβ+; NG2+ (yellow) pericytes over total pericytes counted per field. n = 5 per time point except P5 (n = 3). LV, lateral ventricles; E, embryonic; P, postnatal. Scale bar, 50 µm

Article Snippet: Samples were then immunostained in PBLEC (5% Triton X- 100, 1 M MgCl2, 1 M CaCl2, and 1 M MnCl2 in 1X PBS) overnight at 4°C with Biotinylated IB4 (1:50; Vector Laboratories; Cat# BAF1042) and/or the following antibodies: goat anti- PDGFRβ (1:100; R&D Systems; Cat# BAF1042), rabbit anti- NG2 (1:700; Millipore; Cat# AB5320), rabbit anti- Desmin (1:500; Abcam; Cat# AB15200), anti– αSMA- FITC (1:200; Millipore Sigma, Cat# F3777).

Techniques: Expressing, Staining, Standard Deviation, Comparison

Journal: The FASEB Journal

Article Title: Pericyte dynamics in the mouse germinal matrix angiogenesis

doi: 10.1096/fj.202200120r

Figure Lengend Snippet: FIGURE 5 Desmin-positive pericyte coverage is deficient in the mouse GM. (A,B) High magnification images of the developing cortex (CTX) and the germinal matrix/postnatal subventricular zone (GM/SVZ) stained for blood vessels using Isolectin B4 (IB4, blue) and for pericytes with anti-PDGFRβ (red) and anti-Desmin (green).63 Purple dashed lines mark the CTX, and green dashed lines highlight the GM/SVZ regions. Cropped individual images (white solid boxes) of the endothelium and pericytes are displayed below each timepoint to better visualize the overlap of Desmin and PDGFRβ expression (A′,B′). Additional cropped images (dashed white boxes) are displayed in B″ to visualize reduced Desmin-positive pericyte coverage of the GM blood vessels. (C) Quantification of Desmin-positive pericyte coverage reveals significantly reduced coverage at E16.5 and E18.5 in the GM and at P5 in the SVZ. Scatter dot plot shows mean values with standard deviation. n = 5 per time point except P5 (n = 3). Data was analyzed using a two-way anova with Bonferroni's multiple comparison test. **p < .01, ***p < .001. (D) Quantification of Desmin+; PDGFRβ− (gray), PDGFRβ+; Desmin− (red) and PDGFRβ+; Desmin+ (yellow) pericytes over total pericytes counted per field. n = 5 per time point except P5 (n = 3). LV, lateral ventricles; E, embryonic; P, postnatal. Scale bar, 50 µm

Article Snippet: Samples were then immunostained in PBLEC (5% Triton X- 100, 1 M MgCl2, 1 M CaCl2, and 1 M MnCl2 in 1X PBS) overnight at 4°C with Biotinylated IB4 (1:50; Vector Laboratories; Cat# BAF1042) and/or the following antibodies: goat anti- PDGFRβ (1:100; R&D Systems; Cat# BAF1042), rabbit anti- NG2 (1:700; Millipore; Cat# AB5320), rabbit anti- Desmin (1:500; Abcam; Cat# AB15200), anti– αSMA- FITC (1:200; Millipore Sigma, Cat# F3777).

Techniques: Staining, Expressing, Standard Deviation, Comparison