af385  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Expression of Lumican and Osteopontin in Perivascular Areas of the Glioblastoma Peritumoral Niche and Its Value for Prognosis

doi: 10.3390/ijms26010192

Figure Lengend Snippet: CMA activity in PCs correlates with patient survival. ( A ) Representative images of peritumoral areas according to patient classification showing co-localization of puncta pattern expression of LAMP-2A protein (dark brown) with the PC marker α-SMA (pink) in microvessels of GB patients. Samples were classified as severe (highest α-SMA/LAMP-2A co-localization), moderate or mild (basal co-localization) according to the histological evaluation. A1 to A3 shows magnifications of microvessels (v). LAMP-2A co-localizationwith α-SMA + cells is marked with arrows. Scale bar: 100 µm. ( B ) Representative images of PCs marked with PDGFRβ (in red) in microvessels (v) showing co-localization of puncta pattern expression of LAMP-2A protein (in green) in peritumoral areas of severity classified GB patients. B1 to B3 show magnifications of PDGFRβ + cells. Positive co-localization (in yellow) is marked with arrows. Nuclei were stained with DAPI (blue). Scale bar: 100 µm. ( C ) Overall survival shown by Kaplan–Meier curves of the severity classification related to CMA activity in PCs; * p < 0.05: difference between mild and severe; # p < 0.05: difference between mild and moderate. ( D ) Age distribution by gender of the cohort of GB patients; **** p < 0.0001; ns indicates no significance. ( E ) Severity classification related to gender in the age range 30–65 years in the cohort of GB patients; **** p < 0.0001; ns indicates no significance.

Article Snippet: For fluorescent double-labeling of patient’s samples, goat anti-platelet-derived growth factor receptor beta (PDGFRβ; 1/250; BAF1042, R&D Systems, Barcelona, Spain), rabbit anti-LAMP-2A (1/1000; 51-2200, Invitrogen, Waltham, MA, USA) and mouse anti-RGS5 (1/150; MA5-25584, Invitrogen, Waltham, MA, USA) antibodies were used.

Techniques: Activity Assay, Expressing, Marker, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Interleukin‐3 Modulates Macrophage Phagocytic Activity and Promotes Spinal Cord Injury Repair

doi: 10.1111/cns.70181

Figure Lengend Snippet: IL‐3 is specifically expressed in astrocytes at 14 dpi. (A) Immunofluorescent staining of CD31 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (B) Immunofluorescent staining of PDGFRβ (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (C) Immunofluorescent staining of CD68 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (D) Immunofluorescent staining of NG2 (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. (E) Immunofluorescent staining of GFAP (green), IL‐3 (red), and nuclei (blue) in sagittal sections at 14 dpi. The region of interest (ROI) represents the boxed region on the left. Asterisks indicate the injured core. Scale bars: 200 μm (left panel) and 20 μm (right panel). n = 3 animals per group. (F) Quantification of the proportion of IL‐3 + CD31 + cells in CD31 + cells, IL‐3 + PDGFRβ + cells in PDGFRβ + cells, IL‐3 + CD68 + cells in CD68 + cells, IL‐3 + NG2 + cells in NG2 + cells, or IL‐3 + GFAP + cells in GFAP + cells at 14 dpi. ND, no determined, ** p < 0.01; **** p < 0.001 by one‐way ANOVA followed by Tukey's post hoc test.

Article Snippet: The primary antibodies used in this study were as follows: rat anti‐IL‐3 (1:10, 503902, Biolegend), rabbit anti‐IL‐3Rα (1:100, 141039, US Biological), goat anti‐CD31 (1:200, AF3625, R&D Systems), rat anti‐F4/80 (1:100, 14‐4801‐82, Invitrogen), goat anti‐PDGFRβ (1:100, AF1042‐SP, R&D Systems), rat anti‐GFAP (1:200, 13‐0300, Invitrogen), rabbit anti‐GFAP (1:100, 16825‐1‐AP, Proteintech), goat‐Iba1 (1:200, NB100‐1028, Novus), rat anti‐CD68 (1:300, MCA1957, AbD Serotec), goat anti‐5‐HT (1:5000, #20080, Immunostar), and rabbit anti‐NeuN (1:500, ab177487, Abcam).

Techniques: Staining

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