Journal: The Journal of Neuroscience

Article Title: Circulating Insulin-Like Growth Factor I Mediates Effects of Exercise on the Brain

doi: 10.1523/JNEUROSCI.20-08-02926.2000

Figure Lengend Snippet: Physical exercise and intracarotid injection of IGF-I produce similar effects in the brain. A , The same brain areas show labeling of neurons with IGF-I after treadmill running ( a–c ) and intracarotid injection of IGF-I ( d–f ). Three representative areas are shown. Nonexercised, saline-injected rats show almost undetectable brain IGF-I staining ( B ). Str , Striatum; Cx , cerebral cortex; RN , red nucleus. Biotinylated anti-rabbit IgG followed by Cy3-streptavidin was used after incubation with a polyclonal anti-IGF-I antibody. B , Digoxigenin ( DIG ) and IGF-I colocalize within the same neurons after intracarotid injection of DIG–IGF-I. A representative field in the brainstem is shown. a , Low magnification (10×) of IGF-I staining in the inferior olive nucleus ( IO ) of a saline-injected rat. Note the absence of signal. Inset , Higher magnification (40×) of the IO field. b , The same field showing IGF-I staining in an IGF-I-injected rat. Inset , High magnification showing IGF-I-positive cells. c , High magnification (40×) of IO neurons stained with a monoclonal anti-DIG antibody ( green ). d , The same field stained with a polyclonal anti-IGF-I antibody ( red ). e , Colocalization of DIG and IGF-I within the same IO neurons. Scale bars: a , b , 500 μm; c – e , 50 μm. Primary antibody incubation was followed by an anti-rabbit Cy2 and anti-mouse Cy5, respectively. C , Exercise or intracarotid injection of IGF-I elicits a similar pattern of increased c-Fos staining throughout the brain. Only the piriform cortex ( Pir ) is shown as a representative area. a , Control animals show no c-Fos staining. b , c-Fos staining after 1 hr of intracarotid injection of IGF-I. c , c-Fos staining after 1 hr of running. Scale bar ( a – c ): 500 μm. d , Higher magnification of the field in c showing nuclear localization of the c-Fos signal. Scale bar, 50 μm. Arrows indicate immunoreactive cells. A monoclonal anti-c-Fos antibody followed by a biotinylated anti-mouse IgG and Cy3-streptavidin was used. D , Blockade of the exercise-induced capture of IGF-I by brain cells results in absence of c-Fos labeling after exercise. a , IGF-I labeling in the hippocampus of a rat that ran for 1 hr. c , Chronic intracerebroventricular delivery of a combination of an anti-IGF-I antibody and an IGF-I receptor antagonist results in absence of IGF-I staining after 1 hr of running exercise. Scale bar ( a , c ): 50 μm. b , c-Fos staining is induced in the hippocampus by 1 hr of running. c , No c-Fos labeling is seen in exercised animals in which brain uptake of IGF-I is blocked. Scale bar ( b , d ): 500 μm. The hippocampus is shown as a representative area, but absence of labeling for IGF-I and c-Fos was found in all brain areas. E , Expression of BDNF in the hippocampus is increased by running and by intracarotid injection of IGF-I. Control: background BDNF RNA staining in brain slices incubated with excess unlabeled probe. Saline: animals injected with saline show weak BDNF expression in the hippocampus. Exercise: running induces increased expression of BDNF in the hippocampus. IGF-I: injection of IGF-I produces a similar increase in hippocampal expression of BDNF. Cx , Cortex; Hy , hippocampus.

Article Snippet: The secondary antibodies that were used were biotinylated goat anti-mouse IgG (1:1000) (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit IgG (1:250–1:1000) (Pierce, Rockford, IL).

Techniques: Injection, Labeling, Staining, Incubation, Expressing

Journal: Nature immunology

Article Title: IgG3 regulates tissue-like memory B cells in HIV-infected individuals

doi: 10.1038/s41590-018-0180-5

Figure Lengend Snippet: IgG3–IgM co-localization on IgG3+IgM+ B cells. a , Imaging cytometry (top right) of CD20+ B cells from a chronically infected HIV-viremic individual, gated (left) on populations with different levels of IgG3 and IgM (arrows at left); below, frequency of IgG3–IgM co-localization, based on quantitative analysis of the relative intensity of two fluorescent images. b , Pearson’s co-localization coefficient ( R ) for various protein pairs (horizontal axis), as measured by TIRF microscopy of B cells from a chronically infected HIV-viremic individual with high-intensity IgG3 on IgM + B cells (H-) and one with low-intensity IgG3 on IgM + B cells (L-), stained for tIgG (with polyclonal anti-IgG), IgG3, IgM and CD45; R values were calculated pairwise for each marker and for each source of cells. c , R values for IgG3–IgM co-localization on the TLM B cells and naive B cells (horizontal axis) of a chronically infected HIV-viremic individual with high-intensity IgG3 on IgM + B cells. Results in b , c are presented as ‘box and whiskers’ plots (middle line, mean; box limits, inter-quartile distance; extended lines, Tukey’s method); in parentheses (above horizontal axis), number of cells imaged for each analysis. **** P

Article Snippet: Membranes were sequentially incubated with goat anti-IgM-HRP (Southern Biotech; Cat# 2020–05) or primary antibody rabbit anti-C1q (Novus; Cat# H00000712-D01P), mouse anti-CRP (R & D Systems; clone 232026), rabbit anti-CD32 (GeneTex; Cat# GTX133371) or rabbit anti- IgG3 (Abcam; clone EPR4419), followed by the secondary antibody, goat anti-rabbit-HRP (Bio-Rad; Cat# 1662408EDU) or goat anti-mouse-HRP (Bio-Rad; Cat# 1000450), diluted in 4% milk powder in 0.1% Tween in PBS.

Techniques: Imaging, Cytometry, Infection, Microscopy, Staining, Marker

Journal: Nature immunology

Article Title: IgG3 regulates tissue-like memory B cells in HIV-infected individuals

doi: 10.1038/s41590-018-0180-5

Figure Lengend Snippet: IgG3+IgM+ B cells have reduced responses to BCR stimulation. a , Flow cytometry (top) of CD20-gated B cells of a chronically infected HIV-viremic individual, showing expression of CD27 and CD21 (far left) and the IgG3 and IgD profiles of the corresponding TLM B cell and naive B cell populations (middle and right); below, calcium uptake by IgG3 − IgD + and IgG3 + IgD + (far left margin) TLM B cells (left) and naive B cells (right) before (Unstimulated) and after (Anti-IgM) 2 min of incubation with F(ab’) 2 anti-IgM; the peak (number in plots) is the ratio of bound fluorescent Ca2 + indicator dye Indo-1 to unbound Indo-1. Data are representative of nine experiments. b , Calcium uptake by IgG3 + IgD + TLM B cells (TLM G3+D+), IgG3 − IgD + TLM B cells (TLM G3 − D + ), IgG3 + IgD + naive B cells (N G3+D+) and IgG3 − IgD + naive B cells (N G3 − D + ) from chronically infected HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM + B cells, presented as the peak ratio (as in a ) after incubation with anti-IgM relative to that ratio in unstimulated cells (‘fold’ values). c , d , Quantification of phosphorylated (p-) Syk, PLC-γ2 and Btk (measured by flow cytometry) in B cell populations as in b (horizontal axis) after 2 min of incubation with F(ab’) 2 anti-IgM ( c ) or in the absence of such incubation with anti-IgM ( d ); results in c after incubation with anti-IgM are presented relative to baseline (‘fold’ values). Each symbol ( b – d ) represents an individual subject ( n = 9 ( b ) or n = 11 ( c , d )); small horizontal lines indicate the median. * P

Article Snippet: Membranes were sequentially incubated with goat anti-IgM-HRP (Southern Biotech; Cat# 2020–05) or primary antibody rabbit anti-C1q (Novus; Cat# H00000712-D01P), mouse anti-CRP (R & D Systems; clone 232026), rabbit anti-CD32 (GeneTex; Cat# GTX133371) or rabbit anti- IgG3 (Abcam; clone EPR4419), followed by the secondary antibody, goat anti-rabbit-HRP (Bio-Rad; Cat# 1662408EDU) or goat anti-mouse-HRP (Bio-Rad; Cat# 1000450), diluted in 4% milk powder in 0.1% Tween in PBS.

Techniques: Flow Cytometry, Cytometry, Infection, Expressing, Incubation, Planar Chromatography

Journal: Nature immunology

Article Title: IgG3 regulates tissue-like memory B cells in HIV-infected individuals

doi: 10.1038/s41590-018-0180-5

Figure Lengend Snippet: Soluble IgG3 binds to IgM-expressing B cells of HIV-viremic individuals in vivo. a,b, Flow cytometry of B cells isolated from the peripheral blood of HIV-negative, HIV-aviremic and HIV-viremic individuals (above plots), CD20 gated (to exclude plasmablasts, which typically express CD19 but not CD20), and stained for IgG3 and tIgG ( a ) or IgM ( b ). Numbers in quadrants indicate percent cells in each throughout. c , Flow cytometry showing immunoglobulin light-chain expression (middle and right) on IgG3 + IgM + and combined single immunoglobulin-positive (Ig + ) B cells of an HIV-viremic individual. d , Flow cytometry showing staining for tIgG, IgG1, IgG3 and IgM of B cells obtained from an HIV-viremic individual and treated with trypsin (bottom row) or not (top row) for 5 min at 37°C. e , Expression of immunoglobulin-encoding mRNA (key) by B cells sorted by the surface expression as tIgG + IgM − , tIgG − IgM + or IgG3 + IgM + (horizontal axis); band intensity was normalized to that of control mRNA encoding β -actin. Data are representative of 83 experiments (HIV-negative), 17 experiments (HIV-aviremic) or 75 experiments (HIV-viremic) ( a,b ), 17 ( c ) or 13 ( d ) experiments, or three independent experiments ( e ).

Article Snippet: Membranes were sequentially incubated with goat anti-IgM-HRP (Southern Biotech; Cat# 2020–05) or primary antibody rabbit anti-C1q (Novus; Cat# H00000712-D01P), mouse anti-CRP (R & D Systems; clone 232026), rabbit anti-CD32 (GeneTex; Cat# GTX133371) or rabbit anti- IgG3 (Abcam; clone EPR4419), followed by the secondary antibody, goat anti-rabbit-HRP (Bio-Rad; Cat# 1662408EDU) or goat anti-mouse-HRP (Bio-Rad; Cat# 1000450), diluted in 4% milk powder in 0.1% Tween in PBS.

Techniques: Expressing, In Vivo, Flow Cytometry, Cytometry, Isolation, Staining

Journal: Infection and Immunity

Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

doi: 10.1128/IAI.00410-07

Figure Lengend Snippet: Immunolocalization of LipY(ΔPE) at the surface of M. bovis BCG. Immunolabelings were performed on whole M. bovis BCG carrying pMV261:: lipY (Δ PE ), deposited on EM Formvar-coated nickel grids prior to labeling (A and B) or onto prefixed bacteria labeled in suspension prior to processing for conventional EM (C to F). In both cases, bacteria were sequentially exposed to rat anti-LipY(ΔPE) antibodies, rabbit anti-rat IgG, and PAO. (A) Whole bacteria on grids exposed to specific antibody, followed by rabbit anti-rat IgG and PAO: the gold particles (300 to 400 per bacterium) are distributed throughout the bacterial surface (arrows). (B) Whole bacteria on grids exposed to rabbit anti-rat IgG and PAO only as a control: bacteria display at most 40 gold particles on their surface (arrow). (C to E) Thin sections of bacteria exposed to specific antibody, followed by rabbit anti-rat IgG and PAO. (C) The outermost layer of the cell wall is labeled (arrow). This is particularly obvious in the enlarged view (D), where the peptidoglycan layer (PG), the thin electron-translucent layer (ETL), and the outermost fibrillar layer (OL) are clearly visible. (E) When the OL has been shed, bacteria are not labeled. (F) Thin sections of bacteria exposed to preimmune rat serum, followed by rabbit anti-rat IgG and PAO: bacteria are not labeled even when the OL is present. Bars in panels A and B = 0.5 μm; Bars in panels C, E, and F = 0.25 μm; Bar in panel D = 0.1 μm.

Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

Techniques: Labeling

Journal: Infection and Immunity

Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

doi: 10.1128/IAI.00410-07

Figure Lengend Snippet: Localization of LipY and LipY(ΔPE) in M. bovis BCG. (A) Subcellular localization of LipY and LipY(ΔPE) in M. bovis BCG strains. Cultures were lysed and fractionated to separate the cytoplasm (Cy) from the cell wall (CW). Equal amounts of proteins (10 μg) of each fraction were subjected to SDS-PAGE, electroblotted onto a nitrocellulose membrane, and probed with either rat anti-LipY(ΔPE) antiserum (top), monoclonal anti-KatG antibodies (middle), or rabbit anti-OmpATb antiserum (bottom). (B) EM immunolocalization of LipY. Thin sections of cryosubstituted M. bovis BCG LipY(ΔPE) were sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG for 1 h, and (iv) PAO for 30 min. Intracytoplasmic labeling is indicated by arrows and surface labeling by arrowheads. Bar = 0.5 μm.

Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

Techniques: SDS Page, Incubation, Labeling

Journal: Infection and Immunity

Article Title: Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY ▿

doi: 10.1128/IAI.00410-07

Figure Lengend Snippet: Specific anti-LipY humoral responses in M. tuberculosis -infected patients as opposed to healthy controls. (A) Purification of the recombinant LipY and LipY(ΔPE) proteins expressed in M. smegmatis carrying either pSD26:: lipY or pSD26:: lipY (Δ PE ). Proteins were extracted from M. smegmatis cell wall preparations, excised from preparative polyacrylamide gels, and then electroeluted to obtain pure proteins. (B) ELISA reactivities of IgG and IgM anti-LipY and anti-LipY(ΔPE) antibodies were assayed in sera of either M. tuberculosis -infected group 1 adult patients or healthy controls (HC) ( n = 44 for healthy controls; n = 69 for patients). (C and D) ELISA reactivities of anti-LipY and anti-LipY(ΔPE) antibodies in two different categories of infected children. (C) IgG and IgM reactivities of sera from recently M. tuberculosis -infected children and from healthy controls ( n = 12 for healthy controls; n = 30 for patients). (D) IgG reactivities for patients with extrapulmonary TB ( n = 12 for healthy controls; n = 27 for patients).

Article Snippet: Thin sections of even thickness (70 nm) were collected on nickel grids and sequentially incubated on drops of (i) PBS containing 5% BSA and 0.1% Tween 20 for 15 min, (ii) rat anti-LipY(ΔPE) antibody for 2 h, (iii) rabbit anti-rat IgG (Dako) for 1 h, and (iv) protein A coupled to gold particles of 5 nm in diameter (PAO) (University of Utrecht, Utrecht, The Netherlands) for 30 min. Antibodies and PAO were diluted in PBS containing 5% BSA and 0.1% Tween 20.

Techniques: Infection, Purification, Recombinant, Enzyme-linked Immunosorbent Assay