Journal: Cell reports

Article Title: Inducible deletion of DGAT1 and 2 from microglia exacerbates neurodegeneration and endolysosomal lipid accumulation in male PS19 mice

doi: 10.1016/j.celrep.2025.116841

Figure Lengend Snippet: Representative images and percent area densities of (A) CD68, (B) Iba1, (C) Gfap, (D) Clec7a, and (E) Tmem119 in the hippocampus and entorhinal/piriform cortical area (EC/PC area) of 9.5-month-old male PCfD mice treated with OIL or TAM at 3 months of age ( n = 16–20 per group). Scale bar: 500 μm. Mann-Whitney tests were used to determine statistical significance (CD68 hippocampus, p = 0.0222). Data are represented as the mean ± SEM. * p < 0.05.

Article Snippet: The primary antibodies were diluted as follows: rabbit- anti -Iba1 (FUJIFILM Wako, 019–19741, 1:500), rat- anti -CD68 (BioRad, MCA1957, 1:500), and guinea pig anti-Plin2 (Progen, GP40, 1:200).

Techniques: MANN-WHITNEY

Journal: Cell reports

Article Title: Inducible deletion of DGAT1 and 2 from microglia exacerbates neurodegeneration and endolysosomal lipid accumulation in male PS19 mice

doi: 10.1016/j.celrep.2025.116841

Figure Lengend Snippet: (A) Left: illustration showing approximate locations of the z stacks acquired for the neutral lipid imaging experiments. This image was generated using BioRender. Right: representative 3D full-thickness Imaris renderings of Iba1, CD68, and BODIPY co-stains of PCfD TAM and OIL hippocampi. All channels were masked outside of Iba1 surfaces to promote visualization of cells throughout the entire stack. Scale bar: 25 μm. Inset scale bar: 10 μm. (B) Quantification of Iba1-CD68-BODIPY colocalized volumes per total Iba1 volume. Significance was determined with a Mann-Whitney test ( n = 17–20 per group, p = 0.015). Data are represented as the mean ± SEM. (C) Volcano plot of lipid species altered between PCfD TAM and OIL groups. Statistically significantly altered species are colored ( n = 10 per group). (D) Heatmap of lipid species extracted from bulk posterior cortices of 9.5-month-old male mice via liquid chromatography-mass spectrometry (LC-MS). * p < 0.05.

Article Snippet: The primary antibodies were diluted as follows: rabbit- anti -Iba1 (FUJIFILM Wako, 019–19741, 1:500), rat- anti -CD68 (BioRad, MCA1957, 1:500), and guinea pig anti-Plin2 (Progen, GP40, 1:200).

Techniques: Imaging, Generated, MANN-WHITNEY, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Cell reports

Article Title: Inducible deletion of DGAT1 and 2 from microglia exacerbates neurodegeneration and endolysosomal lipid accumulation in male PS19 mice

doi: 10.1016/j.celrep.2025.116841

Figure Lengend Snippet: (A) Volcano plot of lipid species altered between CD11b high /CD45 int brain myeloid cells from male and female 9.5-month-old WT and PS19 mice (without floxed DGAT alleles) previously gavaged with tamoxifen at 3 months of age. Statistically significantly altered species are colored ( n = 13–14 per group, sexes combined). (B) Representative high-resolution (100×) image of Iba1, BODIPY, CD68, and Plin2 immunofluorescent stains in the hippocampus of a 9-month-old male standard PS19 mouse without floxed DGAT alleles or any treatments. Scale bar: 10 μm. We observed this general staining pattern at 63× in dozens of PS19 and WT mice of multiple strain backgrounds, including PCfD mice treated with TAM and OIL. (C) Representative TEM images of brain myeloid cells from dentate gyrus granule cell layers of 10-month-old male WT and PS19 mice. Myeloid cells were identified by the presence of heterochromatin around the edges of irregularly shaped nuclei. Left: low-magnification images (3,000×, scale bar: 2 μm). Right: high-magnification views (15,000×, scale bar: 400 nm) of lipid-laden lysosomes highlighted in the dashed squares. The red arrows indicate single lysosomal membranes, and the white arrows indicate double lysosomal membranes associated with lipid accumulation. Images are representative of what we have observed from reviewing 6–8 images per sample from 3 PS19 and 2 WT male mice.

Article Snippet: The primary antibodies were diluted as follows: rabbit- anti -Iba1 (FUJIFILM Wako, 019–19741, 1:500), rat- anti -CD68 (BioRad, MCA1957, 1:500), and guinea pig anti-Plin2 (Progen, GP40, 1:200).

Techniques: Staining

Journal: Biomedicines

Article Title: Resveratrol and Its Analogue 4,4′-Dihydroxy-trans-stilbene Inhibit Lewis Lung Carcinoma Growth In Vivo through Apoptosis, Autophagy and Modulation of the Tumour Microenvironment in a Murine Model

doi: 10.3390/biomedicines10081784

Figure Lengend Snippet: ( a , b ) Representative images of CD163 immunostaining in vehicle, DHS, and RSV tumour masses (scale bars = 100 μm; 20× magnification ( a )) and in vehicle sample (scale bar 50 μm; 100× magnification ( b )); ( c ) Quantitative analysis of CD163, CD68 and CD3 positive cells/sample (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The primary antibodies used for immunohistochemistry (IHC) were the following: anti-PCNA mouse monoclonal antibody (PC10, Dako, Agilent Technologies, Santa Clara, CA, USA, 1:200), anti-CD31 rat monoclonal antibody (e-Bioscience, Thermo Fisher Scientific, Waltham, MA, USA, 1:400), anti-CD163 RabMab rabbit mAb (Abcam, Cambridge, UK, Clone:EPR19518, 1:500), anti-CD3 rabbit polyclonal antibody (Origene, Rockville, MD, USA, TA354250, 1:200) and anti-CD68 rat monoclonal antibody (Origene, Rockville, MD, USA, SM1550PS, 1:200) were used.

Techniques: Immunostaining

Journal: PLoS ONE

Article Title: Effects of prenatal low protein and postnatal high fat diets on visceral adipose tissue macrophage phenotypes and IL-6 expression in Sprague Dawley rat offspring

doi: 10.1371/journal.pone.0169581

Figure Lengend Snippet: A-I) ATMs were stained with anti-CD68 antibody. The number of ATMs was counted per 1,000 adipocytes area. Three randomly selected areas were counted for each rat. Average number of the ATMs was used for further statistical analysis. Number of CD68 positive cells in each group is normalized to the number of CD68 + macrophages in NP+NE group. Data are presented as mean ± SEM, n = 6–9.”#” P<0.05 compared to NE; “*” P<0.05 compared to NP. E, F, G, and H are enlargements of the framed fields of A, B, C, and D, respectively. Magnificence of A-D: 200X; E-H 400X.

Article Snippet: Then SVCs were incubated with anti-rat CD68 FITC (Acris-antibodies, San Diego, CA) for 30 minutes at 4°C in dark and washed with Magnetic Cell Separation (MACS) buffer (Miltenyi Biotec Inc., Auburn, CA).

Techniques: Staining

Journal: PLoS ONE

Article Title: Effects of prenatal low protein and postnatal high fat diets on visceral adipose tissue macrophage phenotypes and IL-6 expression in Sprague Dawley rat offspring

doi: 10.1371/journal.pone.0169581

Figure Lengend Snippet: P value of Two-way ANOVA for LP and HE on ATM phenotype ^a .

Article Snippet: Then SVCs were incubated with anti-rat CD68 FITC (Acris-antibodies, San Diego, CA) for 30 minutes at 4°C in dark and washed with Magnetic Cell Separation (MACS) buffer (Miltenyi Biotec Inc., Auburn, CA).

Techniques: Significance Assay

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: Comparisons between transcriptomic platforms and immunohistochemistry suggest zonation patterns of selected hepatic populations To provide information on the zonation of hepatic populations within hepatic lobules, key cluster markers of each cell type were examined in PC1 and PC2 of each spatial sample. (A) Dot plot indicating the relative expression of marker genes in each population of the snRNA-seq map. The size of the circle indicates the percentage of cells expressing the marker of interest, and the color represents the average expression value of the marker. Expression values of (B) mesenchymal marker Ecm1 (C) cholangiocyte marker Anxa4 (D) myeloid marker Cd163 (E) non-inflammatory myeloid marker Marco (F) myeloid marker Cd68 . Red and dark blue indicate higher and lower expression values in each spot, respectively. (G) Representative spatial distributions of CD68 + cells in the rat liver lobule. Rectangular layers 350 um wide were drawn from the portal tract (layer 1) to the central vein (layer 10) region. Digital images were scanned at 20× magnification. The scale bar represents 100 μm in the full image and 20 μm in the enhanced area. Each rectangular layer is referred to as a region of interest (ROI). (H) Quantification of CD68 + cell densities (#CD68+ cells/layer mm2) in the liver lobule for DA and LEW rats. 30 ROIs were assessed per strain across three animals. A higher number of CD68 + cells were detected near the periportal area. No significant strain-specific differences in the spatial distribution of CD68 + cells were noted. (I) Representative spatial distributions of CD163+ cells in the rat liver lobule. (J) Quantification of CD163+ cell densities (#CD163+ cells/layer mm2) in the liver lobule for DA and LEW rats. The statistical analysis reflects similar strain and zonation patterns as CD68. Statistical significance was determined using a two-way ANOVA followed by Sidak’s multiple comparisons test. (∗: p value <0.05, ∗∗: p value <0.01, ∗∗∗: p value <0.001,∗∗∗∗: p value <0.0001). Data are represented as mean ± SEM. Each dot in H and J represents an ROI region (n = 30). ROI: region of interest, BD: bile duct, CV: central vein, PV: portal vein.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Immunohistochemistry, Expressing, Marker

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: Varimax PCs capture rat hepatic cell identity signatures and strain-specific differences (A) Bar plot representing the feature importance scores (mean decrease Gini impurity) of the top 20 features (varimax factors) of the random forest model trained to predict the strain attributes of the rat hepatic cells. Varimax PC5 and 15 are the most informative features to differentiate cells of each strain from another, which indicates the two factors have captured strain-related variations within the map. (B) A correlation heatmap between the average gene expression of each cluster and the loading scores of varimax factors (capturing the contribution of all genes to a factor). Columns are varimax factors and rows are cell populations. Each cell-type cluster is defined by key marker genes, and dark red or blue indicates that the expression of a marker gene set is positively or negatively correlated, respectively, with a particular varimax factor. A high absolute correlation value indicates a match between a varimax factor and a cell-type cluster. (C) The projection of cells over varimax-1 and 5 indicates that the cells from each strain form distinct clusters over varimax-5. (D) Boxplot indicating the distribution of varimax-5 score over each strain. Cells from DA and LEW strains represent significantly different varimax-5 scores (Wilcoxon-test p value <2.2e-16), indicating that varimax-5 has captured strain differences. (E) The top 10 genes on the top (left table) and bottom (right table) of the varimax-5 loading list mainly contain known hepatocyte markers, indicating that varimax-5 has captured hepatocyte-specific strain differences. Genes with high positive scores (left table) are associated with the DA strain and genes indicating negative loading scores (right table) are LEW-related. The absolute loading scores indicate the contribution of each gene to the corresponding factor. (F) Projection of cells over varimax-1 and 15 indicates that a population of cells from each strain (dotted lines) forms distinct clusters over varimax-15. Annotation of the selected cells indicates that they are mainly from the Marco + myeloid cluster 5. (G) Boxplot indicating the distribution of hepatic cells based on strain over varimax-15. (Wilcoxon-test p value <2.2e-16). The outlier data points (dotted lines) are mainly myeloid cells. (H) The top 10 genes with positive (right table) and negative (left table) varimax-15 loading scores are immune-response related. Genes with positive scores (right table) are associated with the LEW strain, and genes indicating negative loading values (left table) are DA related. The absolute loading scores indicate the contribution of each gene to the corresponding factor. (I) Expression pattern of known myeloid marker genes Marco , Vsig4 , Cd68 , and Lyz2 over UMAP. Dark green represents high expression values. The distribution of general myeloid markers ( Cd68 , Vsig4 ) and non-inflammatory myeloid marker ( Marco ) is consistent with the varimax-15 distribution ( Figure 2 J). (J) The UMAP projection of cells colored based on the varimax-15 score shows the enrichment of varimax-15 over Marco + myeloid population (cluster 5). Darker colors represent higher values of varimax-15 scores. Data are represented as mean ± SEM with each dot representing a single cell. Corrcoef.: correlation coefficient, Var: varimax PC. varimax PCs are referred to as PCs within the main text.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Gene Expression, Marker, Expressing

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet: The inflammatory potential of myeloid cells found in LEW rats is greater than that found in DA rats Myeloid cell inflammatory potential was evaluated after lipopolysaccharide (LPS) stimulation of freshly isolated liver-resident non-parenchymal cells. LPS-induced TNFα secretion was measured via intracellular cytokine staining (ICS). The non-parenchymal liver cell dissociate was obtained via a gentle enzymatic perfusion process and differential centrifugation. The resulting cells were plated in 12 well plates for 3.5 h before being stimulated for 6 h under a concentration of 1 ng/mL of LPS in the presence of 1:1000 concentration of Monensin and Brefeldin. (A) Flow cytometry plots showing the gating strategy for macrophages. (B) Percentage of TNFα + secreting CD68 + CD11b + myeloid cells in the unstimulated control and stimulated conditions of Dark Agouti and Lewis macrophages. (C) Summary graphs of Lewis versus Dark Agouti total TNFα as a percentage of CD68 + CD11b + myeloid, (D) and of the mean fluorescence intensity (MFI) of Lewis vs. Dark Agouti TNFα. (E) Representative flow cytometry plot of TNFα secretion patterns based on ITGAL subpopulations. (F) and summary graph ITGAL expressing CD68 + CD11b + myeloid subpopulations. Plotted are the values from all 4 experimental replicates. Statistical significance for ICS was determined using a non-parametric 2 tailed Mann-Whitney test. (n = 4) (G) Cytometric bead array (LEGENDplex) was performed to quantify the level of cytokines (TNFα, Il-18, CXCL1) on culture supernatants of enriched CD68 + myeloid cells after 24 h of stimulation in various LPS concentration conditions (0, 0.05, 0.1, 1, 10 ng/mL). Three technical replicates were used per animal. Statistical significance of the CBA was determined using a two-way ANOVA and Sidak’s multiple comparisons test (n = 3) Data are represented as mean ± SEM with each dot representing a single animal. (∗: p value <0.05, ∗∗: p value <0.01, ∗∗∗: p value <0.001,∗∗∗∗: p value <0.0001) DA: dark agouti, LEW: lewis, SSC-A: side scatter area, FSC-A: forward scatter area.

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Isolation, Staining, Gentle, Centrifugation, Concentration Assay, Flow Cytometry, Control, Fluorescence, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: A rat liver cell atlas reveals intrahepatic myeloid heterogeneity

doi: 10.1016/j.isci.2023.108213

Figure Lengend Snippet:

Article Snippet: The resulting NPC was stained with a FITC conjugated anti-rat CD68 recombinant antibody (Miltenyi, Clone:REA237).

Techniques: Recombinant, SYBR Green Assay, Staining, Selection, Gene Expression, Software

Journal: Molecular Vision

Article Title: P2X 7 receptor activation may be involved in neuronal loss in the retinal ganglion cell layer after acute elevation of intraocular pressure in rats

doi:

Figure Lengend Snippet: Representative photomicrographs of double immunostaining of the P2X 7 receptor and CD68 in the normal retina and in the retina on days 1, 2, 3, and 7 after intraocular pressure elevation. P2X 7 -positive cells were also stained with anti-CD68 in the ganglion cell layer (GCL). Bar=100 µm.

Article Snippet: For this, mouse Alexa Fluor 488–labeled anti-TUJ1 monoclonal antibody (A488–435L, Covance Research Products, Princeton, NJ) and mouse anti-CD68 (MAB1435, Merck Millipore, Billerica, MA) were used.

Techniques: Double Immunostaining, Staining

Journal: Molecular Vision

Article Title: P2X 7 receptor activation may be involved in neuronal loss in the retinal ganglion cell layer after acute elevation of intraocular pressure in rats

doi:

Figure Lengend Snippet: Representative photomicrographs of double immunostaining of CD68 and TNF-α or interleukin-1β (IL-1 β) in the normal retina and the retina on day 2, with and without treatment of oxidized adenosine triphosphate (OxATP) at 30 µM just after intraocular pressure (IOP) elevation. A: TNF- α, B: IL-1 β. Immunoreactivities of tumor necrosis factor-α (TNF-α) and IL-1β were upregulated in the ganglion cell layer (GCL) and inner plexiform layer (IPL) cells on day 2 after IOP elevation; they were subsequently suppressed by treatment with OxATP. Bar=100 µm.

Article Snippet: For this, mouse Alexa Fluor 488–labeled anti-TUJ1 monoclonal antibody (A488–435L, Covance Research Products, Princeton, NJ) and mouse anti-CD68 (MAB1435, Merck Millipore, Billerica, MA) were used.

Techniques: Double Immunostaining