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: 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

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: 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