Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: White matter microglia/macrophages are activated in the dorsal funiculi of male Plp1 mutant mice. Cervical spinal cord sections from ( A ) mildly-affected P16 rsh ( B ) severely-affected P16 msd ( C ) subclinical P25 Plp1 #66 heterozygous ( D ) moderately-affected P25 Plp1 #66 homozygous mice labeled with antibodies against Iba-1 (red) and CD68 (green). Many Iba-1 positive microglia/macrophages with swollen cell bodies and short processes are labeled with anti-CD68 antibodies in rsh and msd mice, which is indicative of activated cells. The insets in these panels show the canonical activated morphology revealed with anti-Iba-1 antibodies and strong CD68 staining. In contrast, there is little evidence for broad CD68 labeling in Plp1 #66 heterozygous mice in panel C, which is consistent with previous data that immune cells are rarely activated . Activation of microglia/macrophages is apparent for Plp1 #66 homozygous mice in panel D, which exhibit an intermediate level of CD68 labeling. Scale bar: 95 μm, insets 15 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Mutagenesis, Labeling, Staining, Activation Assay

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Proportion of cervical spinal cord Iba-1 + microglia/macrophages in dorsal column, ventral column and lateral funiculus that are activated (CD68 + ).

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques:

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Oligodendrocytes in gray matter from P16 rsh and msd mice do not undergo a UPR and microglia/macrophages exhibit resting morphology. ( A – C ) Iba1 (red) and CD68 (green) antibody labeling in dorsal spinal cord white matter tracts of P16 wild type ( A ) rsh ( B ) and msd ( C ) mice. Although the morphology and CD68 staining is characteristic of resting microglia/macrophages in wild type mice (white arrowheads), these cells are activated in the Plp1 mutants. ( D – F ) In contrast to white matter regions, microglia/macrophages have a resting state phenotype in the adjacent substantia gelatinosa (gray matter) from wild type and mutant mice (black arrowheads). ( G – I ) A major difference between these regions in rsh and msd mice is the relative abundance of oligodendrocytes undergoing an unfolded protein response (UPR), which is evident by the large number of CHOP + cells (green) in white matter (above the dotted line) compared to gray matter (below). We have previously shown that 100% of CHOP + cells in these mutants are oligodendrocytes . Dotted lines mark the white/gray matter boundary. Scale bar in I: 100 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Antibody Labeling, Staining, Mutagenesis

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Activation of rsh and msd microglia/macrophages in optic nerve at P16. ( A ) Longitudinal section from wild type mouse reveals Iba-1 + microglia/macrophages (red) with non-activated morphology (arrowheads). Most of these cells do not express CD68 or express the protein at low levels (green). ( B ) DAPI staining showing the nuclei of the microglia/macrophages in ( A ). ( C , E ) Iba-1 + microglia/macrophages from rsh ( C ) and msd ( E ) mice exhibit an activated morphology with enlarged cell bodies and thickened processes. Most of these cells express CD68 at high levels, which is localized to perinuclear regions. ( D , F ) DAPI staining of the fields in ( C , E ). Scale bar in F: 30 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Activation Assay, Staining

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Expression fold changes of other non-chromosome 17 interferon-induced genes in microarray data for rsh and msd mice.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Expressing, Microarray

Journal: International Journal of Molecular Sciences

Article Title: Multiple Immunostainings with Different Epitope Retrievals—The FOLGAS Protocol

doi: 10.3390/ijms23010223

Figure Lengend Snippet: Secondary antibodies, chromogens, fluorophores and metals. Except DAPI, the fluorophores not directly coupled to antibodies are linked to streptavidin for binding of biotinylated antibodies. Abbreviations: RTU: ready to use, AP: alkaline phosphatase, HRP: horseradish peroxidase. Tb: Terbium, Er: Erbium.

Article Snippet: , 159Tb (conjugated to CD68 clone KP1) , , - , Fluidigm (SKU 201300).

Techniques: Binding Assay

Journal: Toxics

Article Title: Polystyrene Microplastics Postpone APAP-Induced Liver Injury through Impeding Macrophage Polarization

doi: 10.3390/toxics10120792

Figure Lengend Snippet: PS MPs pretreatment exhibited strong macrophage recruitment capacity after APAP injury. ( A ) The changes in CD68 expression were analyzed by Western blotting. Relative ( B ) Ccl2, ( C ) CCR2 and ( D ) Cx3cr-1 mRNA expression ( n ≥ 3). *: p < 0.05, **: p < 0.01 compared to control.

Article Snippet: After 2 h blocking by 5% nonfat milk at room temperature, the membranes were further incubated with primary antibody β-actin (1:2000, Proteintech, Wuhan, China) and CD68 (1:1000, Proteintech) at 4 °C overnight.

Techniques: Expressing, Western Blot, Control

Journal: The British journal of dermatology

Article Title: Collagen deposition in chronic Hidradenitis Suppurativa: Potential role for CD163 + macrophages

doi: 10.1111/bjd.16600

Figure Lengend Snippet: (a) Perilesional and chronic lesional HS tissue sections demonstrate dermal infiltration of inflammatory cells, including macrophages in affected areas (n=15, H&E). (b) Immunohistochemistry demonstrates a CD163+ infiltrate in the dermis of in HS chronic lesional tissue (n=8, 20x, scale bar 100 μm outer panels; 10x, scale bar 250 μm middle panel), especially surrounding hair follicles (black arrowheads, 20x, scale bar 100 μm). (c) Quantification of CD163 protein expression in perilesional and lesional regions (mean ± SD, *p=0.0357, Mann-Whitney U test). (d) Flow cytometry demonstrates a 29.4% increase in CD68+C163+ cells in chronic HS lesions (n=3, pink) compared to normal control tissue (n=2, grey). Live single cells were used to set population gates followed by selecting CD68+ cells. (e) Indirect immunofluorescence for CCL18 in chronic HS lesions (n=5), normal control tissue (n=3) and perilesional tissue (n=3) (10x, scale bar 250 μm). (f) Double indirect immunofluorescence staining for CD163 (red), CCL18 (green), and DAPI for nuclei staining (blue). The images for CD163 and CCL18 were merged (yellow-gold) (n=5, 20x, scale bar 100 μm). (g) HS chronic lesional tissue (n=5) show inflammatory infiltrate (black arrow) adjacent to collagen (yellow asterisks, blue stained material) as well as near a hair follicle (black arrowhead) (10x, scale bar 250 μm, perilesional (n=5), Masson’s trichrome (MT)). (h) Picrosirius Red (PSR) staining shows more organized thick, mature, dense bundles of collagen in perilesional tissue (n=1) and thinner, less mature, loose collagen bundles in the chronic lesional HS tissue (n=2) (white arrows, 20x, scale bar 250 μm). (i) Mixed infiltrate including macrophages (black arrow) and reactive myofibroblasts (black double arrow) are present in chronic lesional tissue, 60x. (j) Schematic of proposed role for macrophages in chronic HS: CD68+CD163+ macrophage release of CCL18 stimulates fibroblast-mediated collagen deposition as seen clinically in chronic HS lesions.

Article Snippet: 12 Normal and lesional HS tissues were subjected to single-cell dissociation via the GentleMACs Octo Dissociator (Miltenyi Biotec, Inc.) and analyzed by flow cytometry to determine surface expression of CD68 (anti-human CD68-PE; 10μl/million cells) and CD163 (anti-human CD163-FITC; 10 μl/million cells) (Miltenyi Biotec, Inc.).

Techniques: Immunohistochemistry, Expressing, MANN-WHITNEY, Flow Cytometry, Control, Immunofluorescence, Staining