Journal: Autophagy

Article Title: Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy

doi: 10.1080/15548627.2022.2080382

Figure Lengend Snippet: Effect of tnfaip2 deletion on diabetes-induced glomerular structural abnormalities, markers of fibrosis, and inflammation. Renal cortex samples from diabetic (DM) and non-diabetic (ND) both WT (ND-WT, DM-WT) and tnfaip2 KO (ND-KO, DM-KO) mice were studied 12 weeks after diabetes onset. (A) Representative periodic acid Schiff (PAS) staining images (magnification 400X, scale bar: 50 µm) and (B) quantification of glomerulosclerosis are shown ( n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (C) Electron microscopy images showing a more prominent mesangial expansion in the glomeruli from DM-KO mice compared to DM-WT mice (magnification 3200X). (D) Immunofluorescence images of glomerular FN1 (fibronectin 1) are shown (magnification 400X, scale bar: 50 µm). (E) Quantification of the percent area of glomerular FN1-positive staining ( n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (F) Tgfb1 mRNA levels were measured by real-time PCR in total renal cortex and corrected for the expression of the housekeeping gene Hprt ( n = 5 mice per group; *p < 0.05 DM-WT vs. ND-WT; DM-KO vs. DM-WT). (G) Glomerular macrophage accrual was evaluated by counting the number of LGALS3/MAC2-positive cells per glomerulus ( n = 6 mice per group; ***p < 0.001 DM-WT vs. ND-WT; DM-KO vs. DM-WT). mRNA levels of Ly6c2 (H), Ccl2 (I), Ccr2 (J), and Tnf (K) were measured in the renal cortex by real time-PCR and the results corrected for the expression of the housekeeping gene Hprt ( n = 5 mice per group; *p < 0.05 DM-WT vs. ND-WT; **p < 0.01 DM-KO vs. DM-WT). (L) Schematic illustration of the protocol used for generating diabetic (DM) tnfaip2 KO chimeric mice. Recipient tnfaip2 KO mice were lethally irradiated, and then reconstituted with bone marrow (BM) from either WT (DM-KO-c WT ) or KO (DM-KO-c KO ) mice. (M) PCR genotyping of peripheral blood cells from chimeric animals. M: marker; NC: no template control. (N) ALB (albumin) excretion rate (AER) was measured after 12 weeks of diabetes in transplanted animals. (*p < 0.05 DM-WT-c WT vs. ND-WT-c WT ; DM-KO-c WT and DM-KO-c KO vs. DM-WT-c WT. (O) Representative images of PAS staining of renal cortex sections from transplanted animals (magnification 400X, scale bar: 50 µm).

Article Snippet: After antigen retrieval and blocking in 3% BSA (Merck Life Science, A6003), 4-μm kidney paraffin sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti-TNFAIP2 (Abcam, ab196659; 1:100), rabbit anti-NPHS2/podocin (Merck Life Science, P0372; 1:200), rat-anti LGALS3/MAC2 (Cederlane, CL8942AP; 1:100), rabbit anti-CDKN1C/P57 (Santa Cruz Biotechnology, sc-8298; 1:100), rabbit anti-SQSTM1/p62 (Abcam, ab91526; 1:100).

Techniques: Staining, Electron Microscopy, Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, Irradiation, Marker

Journal: Cell

Article Title: Microglia maintain structural integrity during fetal brain morphogenesis

doi: 10.1016/j.cell.2024.01.012

Figure Lengend Snippet: Microglia limit the progression of CSA microcavities into large lesions in response to morphogenetic constraints (A) IBA1, Spp1, and Mac2 immunostaining findings indicate that ATM line microcavities (solid arrowheads), visible by Hoechst counterstaining at E14.5 (n = 6, from at least two distinct litters). (B) Transmitted electron microscopy (EM) also reveals the presence of microcavities (solid arrowheads), lined with amoeboid microglia (green pseudo-color), and containing membrane fragments (open arrowheads) (n = 3, from at least two distinct litters). (C and D) Coronal hemisections (C) from control and Emx1 cre/+ ;RhoA fl/fl mice, the latter displaying a periventricular nodular heterotopia (PVNH) visible from E18.5 onward (dotted lines). Hoechst counterstaining shows the absence of CSA lesions in controls and mutants at E15.5 (open arrowheads), a striking CSA lesion (solid arrowheads) in 50% of the E18.5 mutants and in 100% of the mutants at P8 (n controls-E15 = 6, n heterotopia-E15 = 5, n controls-E18 = 8, n heterotopia-E18 = 10, n controls-P8 = 10, n heterotopia-P8 = 15). Quantification in (D) uses values that represent the scoring of lesion severity, scored from 0 to 2, as detailed in Table S2 . (E) Coronal hemisections of E15.5 brains from Brn4 cre/+ ; Wnt3a dta/+ embryos show an ablation of the thalamus (white asterisk) and a global change in brain shape but no marked impact on the CSA (n controls = 4, n thalamusdeleted = 5). (F and G) Coronal hemisections (F) of E15. 5 brains from control and Brn4 cre/+ ; Wnt3a dta/+ mice exposed to PLX3397 between E12.5 and E15.5. While PLX3397-exposed controls consistently displayed lesions (open arrowheads), PLX3397-treated mutants exhibited smaller lesions or no lesions (solid arrowheads) despite effective local depletion as assessed by IBA1 staining (n controls = 4, n thalamusdeleted = 5, n PLX3397 = 8, n thalamusdeleted-PLX3397 = 5, from at least two distinct litters). Quantification in (G) uses values that represent the scoring of lesion severity, scored from 0 to 2, as detailed in Table S2 . Graphs show means ± SEM. Mann-Whitney U tests were performed for statistical comparison, ∗ p < 0.05; ∗∗∗ p < 0.001; ns, non significant (p > 0.05). Scale bars: 200 μm (A, left); 100 μm (A and F, high magnifications); 2.5 μm (B); and 500 μm (C, E, and F, low magnification). Am, amygdala; CSA, cortico-striato-amygdalar boundary; PVNH, periventricular nodular heterotopia; Ncx, neocortex; Str, striatum; Th, thalamus. See also Figure S3 .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated at 4°C overnight in the same blocking solution with the following primary antibodies: rat anti-CD68 (1/500; Bio-Rad Cat# MCA1957, RRID: AB_322219 ), rabbit anti-FN1 (1/200; Millipore Cat# AB2033, RRID: AB_2105702 ), goat anti-FOXP2 (1/500; Santa Cruz Biotechnology Cat# sc-21069, RRID: AB_2107124 ), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID: AB_10000240 ), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID: AB_839504 ), rabbit anti-IBA1 (human) (1/500; Abcam Cat# ab178846, RRID: AB_2636859 ), chicken anti-IBA1 (1/500; Synaptic Systems Cat# 234009, RRID: AB_2891282 ), rat anti-Lgals3 (MAC2) (1/1000; CEDARLANE Cat# CL8942AP, RRID: AB_10060357 ), rat anti-L1 (1/100; Millipore Cat# MAB5272, RRID: AB_2133200 ), biotinylated rat anti-LYVE1 (1/200; Thermo Fisher Scientific Cat# 13-0443-82, RRID: AB_1724157 ), rat anti-Myelin Basic Protein (MBP) (1/300; Millipore Cat# MAB386, RRID: AB_94975 ), rat anti-mDectin-1 (CLEC7A) (1/30; InvivoGen Cat# mabg-mdect, RRID: AB_2753143 ), mouse anti-neurofilament marker SMI-312 (1/300; BioLegend Cat# 837904, RRID: AB_2566782 ), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330, RRID: AB_2112934 ), rabbit anti-P2Y12 receptor (1/500; AnaSpec; EGT Group Cat# 55043A, RRID: AB_2298886 ) and goat anti-SPP1 (1/400; R and D Systems Cat# AF808, RRID: AB_2194992 ).

Techniques: Immunostaining, Electron Microscopy, Membrane, Staining, MANN-WHITNEY, Comparison

Journal: Cell

Article Title: Microglia maintain structural integrity during fetal brain morphogenesis

doi: 10.1016/j.cell.2024.01.012

Figure Lengend Snippet: ATM-like microglia are induced by morphogenetic constraints and tissue mechanical lesions (A and B) Coronal E18.5 brain hemisections immunostained with IBA1 and Spp1 showing a marked recruitment of Spp1-expressing microglia at the CSA of Emx1 cre/+ ;RhoA fl/fl mice, with approximately 75% of Spp1-positive CSA microglia in mutants, but 8% of CSA cells detected in controls at this stage (n = 4 at least for each condition, from a minimum of two distinct litters). (C and D) Coronal E15.5 brain hemisections immunostained for IBA1 and Spp1 showing a significantly diminished percentage of CSA microglia expressing Spp1 in E15.5 Brn4 cre/+ ; Wnt3a dta/+ mutant mice compared to controls, despite a conserved number of accumulating cells (n = 4 at least for each condition, from a minimum of two distinct litters). (E) Schematic representation of in utero lesion (IUL) procedure induced by mechanical poking of the neocortex using a glass capillary. (F) Coronal sections through the E14.5 neocortex of control and IUL embryos collected 2.5 h after lesion induction, showing amoeboid Cx3cr1 gfp -positive cells accumulating at the lesion site and the co-expression of Spp1 and Mac2 (solid arrowheads) in IUL embryos but dispersed Cx3cr1 gfp -positive cells and no expression of ATM markers in controls (n controls = 9, n IUL = 8, from at least two distinct litters). Quantification of the percentage of Cx3cr1 gfp - positive cells at the lesion site co-expressing ATM markers (n SPP1 = 4, n MAC2 = 3, n GPNMB = 4 from at least two distinct litters). Graphs show means ± SEM. Mann-Whitney U tests were performed for statistical comparison, ∗ p < 0.05. Scale bars: 200 μm in (A) and 100 μm in (C) and (F). Am, amygdala; CSA, cortico-striato-amygdalar boundary; Ncx, neocortex; Str, striatum.

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated at 4°C overnight in the same blocking solution with the following primary antibodies: rat anti-CD68 (1/500; Bio-Rad Cat# MCA1957, RRID: AB_322219 ), rabbit anti-FN1 (1/200; Millipore Cat# AB2033, RRID: AB_2105702 ), goat anti-FOXP2 (1/500; Santa Cruz Biotechnology Cat# sc-21069, RRID: AB_2107124 ), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID: AB_10000240 ), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID: AB_839504 ), rabbit anti-IBA1 (human) (1/500; Abcam Cat# ab178846, RRID: AB_2636859 ), chicken anti-IBA1 (1/500; Synaptic Systems Cat# 234009, RRID: AB_2891282 ), rat anti-Lgals3 (MAC2) (1/1000; CEDARLANE Cat# CL8942AP, RRID: AB_10060357 ), rat anti-L1 (1/100; Millipore Cat# MAB5272, RRID: AB_2133200 ), biotinylated rat anti-LYVE1 (1/200; Thermo Fisher Scientific Cat# 13-0443-82, RRID: AB_1724157 ), rat anti-Myelin Basic Protein (MBP) (1/300; Millipore Cat# MAB386, RRID: AB_94975 ), rat anti-mDectin-1 (CLEC7A) (1/30; InvivoGen Cat# mabg-mdect, RRID: AB_2753143 ), mouse anti-neurofilament marker SMI-312 (1/300; BioLegend Cat# 837904, RRID: AB_2566782 ), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330, RRID: AB_2112934 ), rabbit anti-P2Y12 receptor (1/500; AnaSpec; EGT Group Cat# 55043A, RRID: AB_2298886 ) and goat anti-SPP1 (1/400; R and D Systems Cat# AF808, RRID: AB_2194992 ).

Techniques: Expressing, Mutagenesis, In Utero, MANN-WHITNEY, Comparison

Journal: Cell

Article Title: Microglia maintain structural integrity during fetal brain morphogenesis

doi: 10.1016/j.cell.2024.01.012

Figure Lengend Snippet: ATM-core factor Spp1 contributes to tissue integrity at the CSA and CSB (A) Coronal hemisections of E14.5 brains stained with Hoechst reveal CSA disruption in 50% of Spp1 −/− mutants (solid arrowheads) compared to controls (open arrowheads) and in 100% of PLX3397-treated embryos (solid arrowheads) (n controls = 18, n Spp1KO = 21, n PLX3397 = 13). Quantification of CSA lesions across models. (B) L1 immunolabeling enables axon visualization (open arrowheads) and midline lesions (solid arrowheads) in approximately 70% of Spp1 −/− mutants compared with 100% in PLX3397-exposed embryos (n controls = 23, n Spp1KO = 20, n PLX3397 = 8). Quantification of CSB lesions across models. (C and D) E14.5 (C) and E18.5 (D) coronal hemisections showing no differences in Mac2 and GPNMB co-expressing microglia at the CSA and CSB of controls (open arrowheads) and Spp1 −/− embryos (n controls = 3, n Spp1KO = 3, at each stage from at least two distinct litters). (E) UMAP visualization of single-cell RNA sequencing (scRNA-seq) data representing macrophage subsets extracted from wild-type (WT) and Spp1 −/− E14.5 and E18.5 embryos colored by annotated clusters (RefinedCellType). (F) Violin plot of normalized and scaled Spp1 expression across annotated clusters between wild-type (WT) (light gray) and Spp1 −/− (KO)(dark gray) mice showing that Spp1 expression is largely restricted to WT ATM (cluster 5). (G) Volcano plot of differentially expressed genes (DEGs) between WT and Spp1 −/− conditions in ATM cells (false discovery rate[FDR]-adjusted p < 0.05 and avgerage_log 2 FC > 0.3). Genes downregulated in Spp1 −/− embryos are displayed in orange, while the upregulated ones are shown in green, and some genes were manually annotated. (H) Bar plots of top Metascape gene set enrichment of DEGs (G) in both WT or Spp1 −/− conditions, highlighting upregulated (green) and downregulated pathways (orange) in Spp1 −/− embryos versus controls, with pathways related to phagocytosis highlighted by a red arrowhead. (I) Brain sections from E15 Cx3cr1 gfp/+ mice showing specific fibronectin 1 (FN1) labeling within GFP- and Mac2-positive ATM microglia at the CSA (performed on brain sections of at least three mice from two distinct litters). (J) High magnification confocal acquisition and 3D cell reconstructions (Imaris software) of immunolabeled sections from E14.5 embryonic brains showing Cx3cr1 gfp -positive CSA microglia with FN1 signal inside cell bodies (performed on brain sections of at least three mice from two distinct litters). (K) Comparison of the percentage of FN1 volume measured (Imaris software) inside individual CSA microglia shows a reduction in Spp1 mutants versus controls (n controls = 17, n Spp1KO = 14, from at least two distinct litters). Graphs show percentages in (A) and (B) and means ± SEM for all others. Fisher’s exact test was performed to compare distributions of cases with lesions in controls, Spp1 −/− , and PLX3397-exposed embryos (A and B), and Mann-Whitney U tests were performed for statistical comparison in all other graphs, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non significant (p > 0.5). Scale bars: 200 μm (A, B low magnification, and D); 100 μm (C and F); 5 μm (G, left); and 2 μm (3D reconstructed cells in F and G). ATM, axon-tract-associated microglia; BAM, border associated macrophages; CC, corpus callosum; CSA, cortico-striato-amygdalar boundary; CSB, cortico-septal boundary; DEGs, differentially expressed genes; Fx, fornix; Intravasc mac, intravascular macrophages; Ncx, neocortex; PVM, perivascular macrophages; Se, Septum; Str, striatum; WT, wild type. See also Figure S4 and Table S3. Genes defining the clusters identified by single-cell transcriptomic analyses on all the sorted cells from wild type and Spp1−/−, related to Figures 5 and S4 , Table S4. Genes defining the clusters identified by single-cell transcriptomic analyses on the macrophages from wild type and Spp1−/−, related to Figure 5 , Table S5. DEGs in ATM in wild type versus Spp1−/−, related to Figure 5 , Table S6. Metascape analyses of ATM in wild type versus Spp1−/−, related to Figure 5 .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated at 4°C overnight in the same blocking solution with the following primary antibodies: rat anti-CD68 (1/500; Bio-Rad Cat# MCA1957, RRID: AB_322219 ), rabbit anti-FN1 (1/200; Millipore Cat# AB2033, RRID: AB_2105702 ), goat anti-FOXP2 (1/500; Santa Cruz Biotechnology Cat# sc-21069, RRID: AB_2107124 ), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID: AB_10000240 ), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID: AB_839504 ), rabbit anti-IBA1 (human) (1/500; Abcam Cat# ab178846, RRID: AB_2636859 ), chicken anti-IBA1 (1/500; Synaptic Systems Cat# 234009, RRID: AB_2891282 ), rat anti-Lgals3 (MAC2) (1/1000; CEDARLANE Cat# CL8942AP, RRID: AB_10060357 ), rat anti-L1 (1/100; Millipore Cat# MAB5272, RRID: AB_2133200 ), biotinylated rat anti-LYVE1 (1/200; Thermo Fisher Scientific Cat# 13-0443-82, RRID: AB_1724157 ), rat anti-Myelin Basic Protein (MBP) (1/300; Millipore Cat# MAB386, RRID: AB_94975 ), rat anti-mDectin-1 (CLEC7A) (1/30; InvivoGen Cat# mabg-mdect, RRID: AB_2753143 ), mouse anti-neurofilament marker SMI-312 (1/300; BioLegend Cat# 837904, RRID: AB_2566782 ), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330, RRID: AB_2112934 ), rabbit anti-P2Y12 receptor (1/500; AnaSpec; EGT Group Cat# 55043A, RRID: AB_2298886 ) and goat anti-SPP1 (1/400; R and D Systems Cat# AF808, RRID: AB_2194992 ).

Techniques: Staining, Disruption, Immunolabeling, Expressing, RNA Sequencing Assay, Labeling, Software, Comparison, MANN-WHITNEY

Journal: Cell

Article Title: Microglia maintain structural integrity during fetal brain morphogenesis

doi: 10.1016/j.cell.2024.01.012

Figure Lengend Snippet: ATM-factor Spp1 contributes to lesion repair (A) Cx3cr1 gfp -positive cells accumulating at the site of lesion closure co-express ATM markers Spp1, Mac2, and GPNMB, as shown and quantified at P3 (n controls = 4; n PLX3397 = 4 for each marker, from at least two distinct litters). (B) Extracellular Spp1 signal, delineated by immunostaining and Hoechst labeling, accumulates at the resorbing CSA at P3. Graphs show the increased signal intensity at the CSA (dotted lines) compared with a mean between signal intensity measured in the surrounding neocortex (dotted lines) and amygdala (dotted lines) in PLX3397-exposed pups versus controls (n controls = 4; n PLX3397 = 4, from two distinct litters). (C) In contrast to controls, Spp1 −/− mutants, and PLX3397-exposed controls, PLX3397-exposed Spp1 −/− mutants reproducibly displayed lesions visible by Hoechst staining (solid versus open arrowheads) (n controls = 7; n Spp1KO = 8; n control-PLX3397 = 11; n Spp1KO-PLX3397 = 7). Values represent the scoring of lesion severity, scored from 0 to 2, as detailed in Table S2 . (D) While CSA IBA1-positive cells co-expressed Mac2 in resorbed PLX3397-treated controls at P7, they also accumulated around the lesions in PLX3397-treated Spp1 mutants, indicating that Spp1 inactivation did not prevent the expression of selected ATM markers (n control-PLX3397 = 3; n Spp1KO-PLX3397 = 7). Graphs show means ± SEM. Mann-Whitney U test was performed for statistical comparison, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, non significant (p > 0.5). Scale bars: 200 μm in (A); 150 μm in (C) and (D); and 100 μm in (B). Am, amygdala; CSA, cortico-striato-amygdalar boundary; Ncx, neocortex; Str, striatum. See also Figure S7 .

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated at 4°C overnight in the same blocking solution with the following primary antibodies: rat anti-CD68 (1/500; Bio-Rad Cat# MCA1957, RRID: AB_322219 ), rabbit anti-FN1 (1/200; Millipore Cat# AB2033, RRID: AB_2105702 ), goat anti-FOXP2 (1/500; Santa Cruz Biotechnology Cat# sc-21069, RRID: AB_2107124 ), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID: AB_10000240 ), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID: AB_839504 ), rabbit anti-IBA1 (human) (1/500; Abcam Cat# ab178846, RRID: AB_2636859 ), chicken anti-IBA1 (1/500; Synaptic Systems Cat# 234009, RRID: AB_2891282 ), rat anti-Lgals3 (MAC2) (1/1000; CEDARLANE Cat# CL8942AP, RRID: AB_10060357 ), rat anti-L1 (1/100; Millipore Cat# MAB5272, RRID: AB_2133200 ), biotinylated rat anti-LYVE1 (1/200; Thermo Fisher Scientific Cat# 13-0443-82, RRID: AB_1724157 ), rat anti-Myelin Basic Protein (MBP) (1/300; Millipore Cat# MAB386, RRID: AB_94975 ), rat anti-mDectin-1 (CLEC7A) (1/30; InvivoGen Cat# mabg-mdect, RRID: AB_2753143 ), mouse anti-neurofilament marker SMI-312 (1/300; BioLegend Cat# 837904, RRID: AB_2566782 ), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330, RRID: AB_2112934 ), rabbit anti-P2Y12 receptor (1/500; AnaSpec; EGT Group Cat# 55043A, RRID: AB_2298886 ) and goat anti-SPP1 (1/400; R and D Systems Cat# AF808, RRID: AB_2194992 ).

Techniques: Marker, Immunostaining, Labeling, Staining, Expressing, MANN-WHITNEY, Comparison

Journal: Cell

Article Title: Microglia maintain structural integrity during fetal brain morphogenesis

doi: 10.1016/j.cell.2024.01.012

Figure Lengend Snippet:

Article Snippet: Slices were first incubated for 1 h at room temperature (RT) in 0.2% Triton X-100, 0.2% Gelatin in PBS (blocking solution), and then incubated at 4°C overnight in the same blocking solution with the following primary antibodies: rat anti-CD68 (1/500; Bio-Rad Cat# MCA1957, RRID: AB_322219 ), rabbit anti-FN1 (1/200; Millipore Cat# AB2033, RRID: AB_2105702 ), goat anti-FOXP2 (1/500; Santa Cruz Biotechnology Cat# sc-21069, RRID: AB_2107124 ), chicken anti-GFP (1/1000; Aves Labs Cat# GFP-1020, RRID: AB_10000240 ), rabbit anti-IBA1 (1/500; FUJIFILM Wako Shibayagi Cat# 019-19741, RRID: AB_839504 ), rabbit anti-IBA1 (human) (1/500; Abcam Cat# ab178846, RRID: AB_2636859 ), chicken anti-IBA1 (1/500; Synaptic Systems Cat# 234009, RRID: AB_2891282 ), rat anti-Lgals3 (MAC2) (1/1000; CEDARLANE Cat# CL8942AP, RRID: AB_10060357 ), rat anti-L1 (1/100; Millipore Cat# MAB5272, RRID: AB_2133200 ), biotinylated rat anti-LYVE1 (1/200; Thermo Fisher Scientific Cat# 13-0443-82, RRID: AB_1724157 ), rat anti-Myelin Basic Protein (MBP) (1/300; Millipore Cat# MAB386, RRID: AB_94975 ), rat anti-mDectin-1 (CLEC7A) (1/30; InvivoGen Cat# mabg-mdect, RRID: AB_2753143 ), mouse anti-neurofilament marker SMI-312 (1/300; BioLegend Cat# 837904, RRID: AB_2566782 ), goat anti-mouse Osteoactivin (GPNMB) (1/200; R and D Systems Cat# AF2330, RRID: AB_2112934 ), rabbit anti-P2Y12 receptor (1/500; AnaSpec; EGT Group Cat# 55043A, RRID: AB_2298886 ) and goat anti-SPP1 (1/400; R and D Systems Cat# AF808, RRID: AB_2194992 ).

Techniques: Marker, Recombinant, Multiplexing, Amplification, Software, Microscopy, Fluorescence, Transmission Assay

Journal: PLoS ONE

Article Title: Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo : Effects of Lipid Lowering by Diet or Atorvastatin

doi: 10.1371/journal.pone.0130898

Figure Lengend Snippet: ( A) Intracellular and extracellular ROS were analyzed in the aortic arch (red area) (B) Smooth muscle cell and macrophage content was quantified in the aortic arch by analyzing sections from 4 different levels. (C) Section stained for macrophages (CD68: blue) and smooth muscle cells (α-actin: red). (D and E) Smooth muscle cell content in lesions correlated with extracellular (E) but not intracellular ROS (D) . (F and G) Macrophage content in lesions correlated with intracellular (F) but not extracellular ROS (G) . Linear regression (n = 25). NS (non significant).

Article Snippet: Double staining was used to simultaneously demonstrate macrophages (rat anti-mouse Mac2, IgG2a, clone CL8942AP, 50 μg/ml, Nordic Biosite AB, Täby, Sweden) and smooth muscle cells (SMC) (polyclonal rabbit anti-human smooth muscle cell actin, 1 μg/ml, ab5694, Abcam, Cambridge, UK).

Techniques: Staining