Journal: Glia

Article Title: Activated microglia do not increase 18 kDa translocator protein ( TSPO ) expression in the multiple sclerosis brain

doi: 10.1002/glia.24052

Figure Lengend Snippet: Antibodies for immunohistochemistry

Article Snippet: CD68 , Rabbit , Atlas Antibody , HPA048982 , 1:4000.

Techniques:

Journal: PLOS One

Article Title: Photobiomodulation therapy increases neural stem cell pool in aged 3xTg-AD mice

doi: 10.1371/journal.pone.0321668

Figure Lengend Snippet: A. Representative confocal images of Aβ plaques (6E10), microglia (Iba1) and CD68 showing diffuse microglial phagocytosis away from plaques in sham compared to localized phagocytosis around plaques in PBM-treated animals. B. Quantitative analysis of total and activated microglia show no overall differences in the neuroinflammatory environment surrounding Aβ plaques. (Unpaired t-test). C. Analysis of the distribution of CD68+ microglia shows a trend of higher concentration of phagocytic cells in contact with plaques, and decreasing phagocytosis away from plaques. (Two-way ANOVA).

Article Snippet: Up to six sections representing different regions of the hippocampus were immunostained with the following primary antibodies: Sox2 (Abcam), doublecortin (DCX, Santa Cruz), calretinin (CR, SWANT), β-amyloid (6E10, Biolegend), phospho-tau (pTau, AT8, Invitrogen), βIII-tubulin (Abcam), Iba1 (Synaptic Systems), and CD68 (Rockland).

Techniques: Concentration Assay

Journal: The American journal of pathology

Article Title: The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

doi: 10.1016/j.ajpath.2017.10.015

Figure Lengend Snippet: Figure 9 Transient versican G1 fragmenteserum-derived hyaluronan-associated proteinehyaluronan (VG1F-SHAP-HA) complex formation in a mouse model of moist wound healing. A: Representative images from wound tissues from the moist wound-healing model are shown. Edematous granulation tissues were observed at day 3 after wounding; however, the granulation tissues were nonedematous and thin at days 1 and 6. The thickness of the granulation tissues is as follows: day 1, 0.77 0.10 mm; day 3, 2.55 0.17 mm; and day 6, 0.82 0.04 mm. B: Tissue specimens were stained with hematoxylin and eosin. C: Isolated tissues were stained by using the indicated probes. The VG1F-SHAP-HA complex was observed on day 3 in specific lesions, but was absent at days 1 and 6. The color of letters indicating molecules represents the color of the stain. The merged images indicate the VG1F-SHAP-HA complex. D: Immunohis- tochemical analysis of wound tissue taken from day 3. The specimen was stained with the indicated probes, and the merged images show VG1F colocalization, as detected by probes for HA, DPEAAE epitope, and macrophages (CD68). The merged images show macrophage/complex colocalization. Data are expressed as means SD (A). n Z 4 independent experiments (A). Scale bars Z 50 mm (BeD). HC, heavy chain.

Article Snippet: A goat pAb against bikunin (sc-21597) was purchased from Santa Cruz Biotechnology Inc. A mouse monoclonal antibody against the macrophage marker CD68 (STJ-6572) was purchased from St. John’s Laboratory Ltd (London, UK).

Techniques: Derivative Assay, Staining, Isolation

Journal: The American journal of pathology

Article Title: The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

doi: 10.1016/j.ajpath.2017.10.015

Figure Lengend Snippet: Figure 11 The versican G1 fragment (VG1F)eserum-derived hyaluronan (HA)eassociated proteineHA complex in human pressure ulcers. A: In human pressure ulcers, edematous wounds (e1 and e2) typically become flat and stabilized (f3 and f4) after appropriate treatments. B: The VG1F-HC complex was detected from the protein extract of wound surfaces shown in A (e1 and e2). The samples are indicated at the top of the figure and correspond to those shown in A. The samples were extracted with 6 mol/L GdnHCl and treated with trypsin and V8 protease (Materials and Methods); separated using SDS-PAGE under nonreducing conditions; and blotted. Incubation with the indicated antibodies revealed the cross-linked heavy chain (HC) and VG1 species (dagger). The bands of cross-linked HC and VG1 species (dagger) disappeared during wound healing. C: Paired samples from edematous or flat wounds were analyzed using Western blotting, and the bands shown in B were quantified. The relative intensities of the bands [VG1-HCs (daggers in B)]/the total polyclonal antibody 6084epositive bands (all VG1) differ significantly (P Z 0.02) between edematous and flat wounds. D: Direct immunofluorescence of wound surface samples obtained from different sites of one individual wound. Samples obtained from the edematous wound (green dashed circle) and flat wound (blue dashed circle) were fixed and incubated with VG1 and HC1 antibodies, as well as biotin-conjugated HAebinding protein, as indicated. A representative result from triplicate experiments with different samples is shown. E: Immunohistochemical analysis of pressure ulcer tissue. The specimen was stained, as indicated. The merged images show VG1F colocalization, as detected by antibodies for DPEAAE epitope, HC1, and macrophage CD68. Data are expressed as means SD (C). n Z 10 (C, each paired sample). Scale bars: 5 mm (D); 100 mm (E).

Article Snippet: A goat pAb against bikunin (sc-21597) was purchased from Santa Cruz Biotechnology Inc. A mouse monoclonal antibody against the macrophage marker CD68 (STJ-6572) was purchased from St. John’s Laboratory Ltd (London, UK).

Techniques: Derivative Assay, SDS Page, Incubation, Western Blot, Immunohistochemical staining, Staining

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 and BNP on macrophage polarization in LPS-activated RAW264.7 cells. ( a ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. ( b , c ) Effect of L2 on M1 and M2 macrophage subtype expression. ( d , e ) Effect of BNP on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 or BNP (0, 0.2, 0.4 and 0.8 ng/mL) and isatin (0.1 mM), added 20 min earlier. ( f ) M2/M1 ratio in LPS-activated RAW264.7 cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 ࢤ cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. L2 corresponding group; †† p < 0.01 vs. group unstimulated with LPS.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Flow Cytometry, Expressing, Immunolabeling, Software

Journal: Toxins

Article Title: Natriuretic-like Peptide Lebetin 2 Mediates M2 Macrophage Polarization in LPS-Activated RAW264.7 Cells in an IL-10-Dependent Manner

doi: 10.3390/toxins15040298

Figure Lengend Snippet: Effect of L2 on macrophage polarization in LPS-activated RAW264.7 cells after interleukin-10 inhibition. ( a , b ) Effect of interlekin-10 (IL-10) on M1 and M2 macrophage subtype expression. Cells were obtained after challenging RAW264.7 cells with LPS for 24 h followed by 48 h treatment with or without exogenous IL-10 (0, 10 and 20 ng/mL). ( c , d ) Effect of L2 on M1 and M2 macrophage subtype expression after IL-10 inhibition. Cells were obtained after challenging RAW264.7 cells with LPS (1 µg/mL) for 24 h followed by 48 h treatment with or without L2 (0, 0.4 or 0.8 ng/mL) and IL-10 inhibitor at 10 µg/mL, added 20 min earlier. ( e ) Representative flow cytometry plots showing the variable macrophage distribution of M1-like macrophages (CD68 + /CD206 − cells, Q1 population) and M2-like macrophages (CD68 + /CD206 + /MRC-1, Q2 population) in control and treated cells. The subtypes of macrophages were identified by analyzing profiles of cell surface markers by FACS. Pro-inflammatory M1 macrophages were identified as CD68 + /CD206 − cells and M2-like macrophages assessed by double immunolabeling of CD68 and CD206/MRC-1 in control and treated cells, and the data were analyzed by BD CellQuestPro software. All results were obtained from duplicate experiments. Data are reported as mean ± SEM. *** p < 0.001 vs. corresponding control LPS group; $ p < 0.05, $$$ p < 0.001 vs. L2 corresponding group.

Article Snippet: RAW264.7 macrophage polarization was detected after treatment with L2 and BNP by flow cytometric profiling of specific surface marker expression, including CD68 for total macrophage population quantification and CD206/MRC-1 for M2-like macrophage quantification using PE Rat Anti-Mouse CD68 and Alexa Fluor ® 647 Rat Anti-Mouse CD206 (BD BioScience, San Jose, CA, USA).

Techniques: Inhibition, Expressing, Flow Cytometry, Immunolabeling, Software

Journal:

Article Title: Haemophilus ducreyi Associates with Phagocytes, Collagen, and Fibrin and Remains Extracellular throughout Infection of Human Volunteers

doi: 10.1128/IAI.69.4.2549-2557.2001

Figure Lengend Snippet: Localization of H. ducreyi in tissues from papular and pustular stages of disease. P, pustule or micropustule. De, dermis. Scale bars represent 100 μm in panels A through F, 50 μm in panels G and I, and 5 μm in panel H. (A) Papule at 48 h doubly stained with polyclonal anti-H. ducreyi antiserum (green) and anticytokeratin MAbs (red). Arrow indicates bacteria (green) in dermis below micropustule. (B) A 48-h papule doubly stained with polyclonal anti-type I collagen (red) and the anti-H. ducreyi MAb BB11 (green). Arrow indicates bacteria colocalizing with collagen (yellow). (C) Section stained as in panel B showing colocalization (yellow) of H. ducreyi and collagen. (D through F) Low-power view of consecutive sections from one pustule stained with polyclonal anti-H. ducreyi antiserum (green) and MAb recognizing PMN elastase (D), CD68 (E), or CD3 (F) (red). (G) Higher-power view of the base of a pustule. Section was stained with polyclonal anti-H. ducreyi antiserum (green), anti-CD68 (red), and the lectin LCA, which stains plasma membranes (blue). Colocalization of anti-CD68 (red) and LCA (blue) on the macrophage cell surface is represented in pink. Note the bacteria between the macrophages and the PMNs of the pustule. (H) High-power view of section stained with polyclonal anti-H. ducreyi antiserum (green) and anti-CD68 (red). Arrow indicates bacteria colocalizing with a macrophage (yellow). (I) Base of a pustule stained with polyclonal anti-H. ducreyi antiserum (green) and anti-CD3 (red). Note that the bacteria do not colocalize with the T cells.

Article Snippet: Sections stained with the anti-CD68 MAb were incubated in Retrieve-All antigen unmasking solution (Signet Pathology Systems, Inc., Dedham, Mass.) for 10 min at 92°C, followed by 10 min at room temperature, and washed three times in PBS prior to the permeabilization step.

Techniques: Staining