Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: CD146 (LSEC) antibody, anti-mouse, FITC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-118-406 RRID: AB_2751506.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: CD146 antibody, anti-human, PE, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-111-322 RRID: AB_2655177.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Journal of Advanced Research

Article Title: Chaperone-mediated autophagy sustains pericyte stemness necessary for brain tissue homeostasis

doi: 10.1016/j.jare.2025.04.015

Figure Lengend Snippet: Donor PC therapy restores CMA in host PCs in damaged tissue. (A) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (green) with a PC marker, α-SMA (red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas (injured) and treated intravenously with control adMSCs and with donor brain WT or KO PCs. Nuclei were stained with DAPI (blue). Scale bar: 100 µm. Right panels show bigger magnification of double positive cells. ( B ) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (in green) with a PC marker, PDGFRβ (in red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas and treated intravenously with control adMSCs and with donor brain WT or KO PCs. Right panels show bigger magnification of double positive cells. Nuclei were stained with DAPI (blue). Scale bars: 100 µm. (C) Representative images showing co-localization of puncta pattern expression of LAMP-2A protein (in green) with a PC marker, CD146 (in red), analyzed in host PCs of microvessels (v) of control mice, in mice brain with lesioned areas and treated intravenously with donor brain PC. Right panels show bigger magnification of double positive cells (Scale bars: 20 µm). Nuclei were stained with DAPI (blue). Scale bars: 100 µm. ( D ) Relative quantification of percentage of PCs in brain microvessels expressing the PC marker α -SMA (n = 40 α -SMA + PCs per condition), PDGFRβ (n = 20 PDGFRβ + PCs per condition), or CD146 (n = 35 CD146 + PCs per condition) and the CMA receptor LAMP-2A (PC/LAMP-2A +/+ ) over total PCs. All data represent mean ± SD obtained from at least, three experiments represented as dots, independently; * p < 0.05; ** p < 0.01; *** p < 0.001; ns indicates no statistical significance (ANOVA with Tukey’s post-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: For fluorescent double labelling, mouse anti-alpha-smooth muscle actin (α-SMA; Abcam, ab7817), goat anti-platelet derived growth factor receptor beta (PDGFRβ; R&D Systems, BAF1042), or rat CD146-APC (Miltenyi Biotec, 130–118-408) antibodies were used in combination with rabbit anti-LAMP-2A (Invitrogen, 51–2200) antibody.

Techniques: Expressing, Marker, Control, Staining, Quantitative Proteomics