Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Antibody combinations used in immunophenotyping

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Details of antibodies used in the study

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques: Concentration Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Characterization of immune cell subtypes in three commonly used mouse strains reveals gender and strain-specific variations

doi: 10.1038/s41374-018-0137-1

Figure Lengend Snippet: Cell suspensions from BM and spleen were isolated from 8-week old mice. The cells were stained with CD11b, F4/80 and CD68 antibodies for macrophages, CD11b and Gr-1 antibodies for iMCs, and CD11b and CD11c antibodies for mDCs. Ly6B was used as negative marker for iMCs. The cells were then subjected to flow cytometry to determine cell-type percentages. (A) iMCs in BM, (n≥7). (B) iMCs in the spleen, (n≥7). (C) Macrophages in BM, (n≥ 6). (D) Macrophages in spleen, (n≥9) (E) mDCs in BM, (n ≥ 10). (F) mDCs in the spleen, (n ≥ 13). Results are shown as scatter plots depicting average cell percentages (percent of live cells). Error bars denote SEM. Each dot represents the value from a single mouse. (♂) and (♀) represent male and female mice, respectively. **p ≤ 0.01, ***p ≤ 0.001, ****p≤0.0001.

Article Snippet: Fixation/Permeabilization Solution Kit with BD Golgi-PlugTM kit and Mouse Foxp3 Buffer Set was used for intracellular staining and purchased from BD Biosciences. table ft1 table-wrap mode="anchored" t5 caption a7 iMC CD11b Vio Blue Gr-1 APC-eFluor780 * Ly6b FITC Macrophages CD11b Vio Blue F4/80 APC CD68 PE mDC CD11b Vio Blue CD11c PE CD80 APC CD86 FITC pDC * CD11b Vio Blue CD11c PE B220 Vio770 Siglec H FITC CD4 T-cells CD3 APC-Cy7 CD4 eFluor450 CD4 T-regs CD3 APC-Cy7 CD4 eFluor450 CD25 PE FoxP3APC CD8 T-cells CD3 APC-Cy7 CD8 FITC B cells B220 Vio770 CD19 PE NK cells CD3 APC-Cy7 NKp46 FITC Neutrophils CD11b Vio Blue Gr-1 APC-eFluor780 Ly6b FITC * F4/80 APC Open in a separate window * negative tor this antibody Antibody combinations used in immunophenotyping table ft1 table-wrap mode="anchored" t5 caption a7 Antibody Fluorochrome Vendor Clone Catalogue # Antibody Concentration (in 100 pL FACS buffer) CD3e APC-Cy7 Fisher Scientific 145–2C11 BDB557596 1 pL CD4 eFluor450 eBioscience GK1.5 48–0041-82 1 pL CD8 FITC eBioscience 53–6.7 11–0081-82 0.6 pL CD11b Vio Blue Miltenyi M1/70.15.11.5 130–097-336 3.75 pL CD11c PE Miltenyi N418 130102545 3.75 pL CD19 PE Miltenyi 6D5 130–102-598 5 pL CD25 PE eBioscience PC61.5 12–0251-81 0.5 pL CD68 PE Miltenyi FA-11 130–102-614 3.75 pL CD80 APC Miltenyi 16–10A1 130–102-584 3.75 pL CD86 FITC Miltenyi PO3.3 130–102-506 5 pL B220 APC-Vio770 Miltenyi RA3–6B2 130–102-267 3.75 pL F4/80 APC Miltenyi REA126 130–102-379 3.75 pL FoxP3 APC eBioscience FJK-16s 17–5773-82 5 pL Gr1 APC-eFluor780 eBioscience RB6–8C5 47–5931-80 1.2 pL Ly6b FITC abcam 7/4 ab53453 0.65 pL NKp46 FITC Miltenyi 29A1.4.9 130–102-300 3.75 pL SiglecH FITC eBioscience eBio440c 11–0333-82 0.5 pL Open in a separate window Details of antibodies used in the study

Techniques: Isolation, Staining, Marker, Flow Cytometry

Journal: Biology of reproduction

Article Title: Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

doi: 10.1095/biolreprod61.3.705

Figure Lengend Snippet: FIG. 1. Light micrographs showing sec- tions of the epididymis of the Brown Nor- way rat stained with an antibody for monocytes-macrophages (ED1). A–C) Cor- pus, D–F) distal cauda. A, D) 3 mo, B, E) 18 mo with numerous spermatozoa in the lumen, C, F) 18 mo with occasional sper- matozoa in the lumen. lu, Lumen; it, inter- tubular space; p, principal cells; b, basal cells; c, clear cells; bm, basement mem- brane. Arrows, ED1-positive cells; aster- isks, halo cells. Scale bar, A–F 5 64 mm.

Article Snippet: Mouse anti-rat ED1 (Serotec USA, Washington, DC) was used at a dilution 1/100; this antibody recognizes a cytoplasmic antigen in monocytes and most macrophages.

Techniques: Staining

Journal: Biology of reproduction

Article Title: Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content.

doi: 10.1095/biolreprod61.3.705

Figure Lengend Snippet: FIG. 5. Light micrographs showing sec- tions of the corpus of the epididymis of Brown Norway rats. A, C) Three mo; B, D) 18 mo. Section stained with an antibody for A and B GST Yf and C and D ED1. Clear arrows, basal cells; dark arrows, ED1-positive cells; other labels as in Fig- ure 1. Scale bar, A–D 5 64 mm.

Article Snippet: Mouse anti-rat ED1 (Serotec USA, Washington, DC) was used at a dilution 1/100; this antibody recognizes a cytoplasmic antigen in monocytes and most macrophages.

Techniques: 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: 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