Structured Review

Agilent technologies icam 1
Immunofluorescence staining with <t>ICAM-1</t> (A) and anti-swine class II antibodies A) Comparison of ICAM1 expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d) native swine kidneys; b and e) non-rejected kidney xenografts; c and f): rejected kidney xenografts. ICAM-1 expression (white arrows) is seen on the peritubular capillaries in pCMV positive kidneys (e and f) while pCMV negative rejected kidney had only a minimal expression of ICAM-1 (c). B) The comparison of class II DR expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d): naive kidneys; b and e): non-rejected kidney grafts, c and f): rejected kidney grafts. Up-regulation of class II on peritubular capillaries and glomeruli (white arrows) in a rejected pCMV negative kidneys (c) as well as pCMV positive rejected kidneys (e and f).
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Images

1) Product Images from "Porcine CMV Infection Is Associated with Early Rejection of Kidney Grafts in a Pig to Baboon Xenotransplantation Model"

Article Title: Porcine CMV Infection Is Associated with Early Rejection of Kidney Grafts in a Pig to Baboon Xenotransplantation Model

Journal: Transplantation

doi: 10.1097/TP.0000000000000232

Immunofluorescence staining with ICAM-1 (A) and anti-swine class II antibodies A) Comparison of ICAM1 expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d) native swine kidneys; b and e) non-rejected kidney xenografts; c and f): rejected kidney xenografts. ICAM-1 expression (white arrows) is seen on the peritubular capillaries in pCMV positive kidneys (e and f) while pCMV negative rejected kidney had only a minimal expression of ICAM-1 (c). B) The comparison of class II DR expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d): naive kidneys; b and e): non-rejected kidney grafts, c and f): rejected kidney grafts. Up-regulation of class II on peritubular capillaries and glomeruli (white arrows) in a rejected pCMV negative kidneys (c) as well as pCMV positive rejected kidneys (e and f).
Figure Legend Snippet: Immunofluorescence staining with ICAM-1 (A) and anti-swine class II antibodies A) Comparison of ICAM1 expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d) native swine kidneys; b and e) non-rejected kidney xenografts; c and f): rejected kidney xenografts. ICAM-1 expression (white arrows) is seen on the peritubular capillaries in pCMV positive kidneys (e and f) while pCMV negative rejected kidney had only a minimal expression of ICAM-1 (c). B) The comparison of class II DR expression in pCMV positive kidneys with negative kidneys following xenotransplantation. a–c were from pCMV negative donors and e-f were from pCMV positive donors: a and d): naive kidneys; b and e): non-rejected kidney grafts, c and f): rejected kidney grafts. Up-regulation of class II on peritubular capillaries and glomeruli (white arrows) in a rejected pCMV negative kidneys (c) as well as pCMV positive rejected kidneys (e and f).

Techniques Used: Immunofluorescence, Staining, Expressing

2) Product Images from "Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury"

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury

Journal: Scientific Reports

doi: 10.1038/s41598-017-03309-4

CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).
Figure Legend Snippet: CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).

Techniques Used: Permeability, Preserving, Immunohistochemistry, Staining, Western Blot, Expressing

Two-photon imaging detection of microglia in CX3CR1-GFP mice and morphometric analysis of cellular morphology in CCI-TBI and anti-ICAM-1/catalase administration. CX3CR1-GFP mice (B6.129P- Cx3cr1 tm1Litt /J, The Jackson Laboratory) were subjected to moderate CCI-TBI with and without anti-ICAM-1/catalase conjugate administration 30 min after impact. No craniectomy (naive) CX3CR1-GFP mice served as control. At 48 hrs following CCI-TBI, subjects were perfusion fixed, brain tissue was collected and subsequently sectioned into 1 mm segments for two-photon imaging. ( A ) Representative single slice images depict microglial ramification in the area of impact for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase mice. Arrowheads point to microglial processes, which are finely ramified in the naive condition and thicken and retract following CCI-TBI. Anti-ICAM-1/catalase attenuates changes in microglia cell body enlargement and process retraction. Scale bar 20 μm. Imaris (Bitplane) software was utilized for 3D reconstruction of GFP-expressing microglia from z-stack images obtained by two-photon microscopy. ( B ) Surface rendering was performed for microglia based on GFP signal threshold to measure cellular surface area ( D ) (only objects with surface area > 1000 μm 2 were analyzed) and sphericity ( E ). ( C ) Imaris FilamentTracer was employed to map microglial processes to quantify changes in cell ramification in CCI-TBI. Microglial total filament length ( F ) and number of filament branching points (complexity, ( G ) and were assessed for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase groups. Data presented at mean ± SD. (Ordinary one-way ANOVA. Surface area: F = 73.12, P
Figure Legend Snippet: Two-photon imaging detection of microglia in CX3CR1-GFP mice and morphometric analysis of cellular morphology in CCI-TBI and anti-ICAM-1/catalase administration. CX3CR1-GFP mice (B6.129P- Cx3cr1 tm1Litt /J, The Jackson Laboratory) were subjected to moderate CCI-TBI with and without anti-ICAM-1/catalase conjugate administration 30 min after impact. No craniectomy (naive) CX3CR1-GFP mice served as control. At 48 hrs following CCI-TBI, subjects were perfusion fixed, brain tissue was collected and subsequently sectioned into 1 mm segments for two-photon imaging. ( A ) Representative single slice images depict microglial ramification in the area of impact for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase mice. Arrowheads point to microglial processes, which are finely ramified in the naive condition and thicken and retract following CCI-TBI. Anti-ICAM-1/catalase attenuates changes in microglia cell body enlargement and process retraction. Scale bar 20 μm. Imaris (Bitplane) software was utilized for 3D reconstruction of GFP-expressing microglia from z-stack images obtained by two-photon microscopy. ( B ) Surface rendering was performed for microglia based on GFP signal threshold to measure cellular surface area ( D ) (only objects with surface area > 1000 μm 2 were analyzed) and sphericity ( E ). ( C ) Imaris FilamentTracer was employed to map microglial processes to quantify changes in cell ramification in CCI-TBI. Microglial total filament length ( F ) and number of filament branching points (complexity, ( G ) and were assessed for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase groups. Data presented at mean ± SD. (Ordinary one-way ANOVA. Surface area: F = 73.12, P

Techniques Used: Imaging, Mouse Assay, Software, Expressing, Microscopy

CCI-TBI results in increased hydrogen peroxide production and 3-nitrotyrosine detection in the ipsilateral cortex area of impact. ( A ) OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit was used to measure hydrogen peroxide levels in fresh mouse cortical tissue ipsilateral to the area of impact 4 hrs following CCI-TBI, CCI-TBI+anti-ICAM-1/catalase, catalase only, and antibody only administration or from naive and sham controls. The naive condition provides basal hydrogen peroxide levels in uninjured cortical tissue which are not significantly different from sham. CCI-TBI results in a nearly 3-fold increase in hydrogen peroxide level detection. Anti-ICAM-1/catalase quenches hydrogen peroxide levels in the area of injury to physiological levels comparable to that of naive and sham, an effect not achieved with anti-ICAM-1 antibody and catalase alone. Immunohistochemical chomogen staining of 3-NT demonstrates increased tyrosine nitration following CCI-TBI ( D ) compared to naive and sham controls ( B and C , respectively). Anti-ICAM-1/catalase significantly decreased 3-NT detection ( E ). 3-NT levels in anti-ICAM-1 antibody and catalase only groups did not significantly differ from CCI-TBI alone ( F and G , respectively). Positive signal detection was pseudo-colored red for better visualization. All images taken at 20X. Scale bar equals 50 microns. ( H ) Quantification of 3-NT signal detection was analyzed by percent area and normalized to total tissue area per image. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons for hydrogen peroxide data F = 25.95, P
Figure Legend Snippet: CCI-TBI results in increased hydrogen peroxide production and 3-nitrotyrosine detection in the ipsilateral cortex area of impact. ( A ) OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit was used to measure hydrogen peroxide levels in fresh mouse cortical tissue ipsilateral to the area of impact 4 hrs following CCI-TBI, CCI-TBI+anti-ICAM-1/catalase, catalase only, and antibody only administration or from naive and sham controls. The naive condition provides basal hydrogen peroxide levels in uninjured cortical tissue which are not significantly different from sham. CCI-TBI results in a nearly 3-fold increase in hydrogen peroxide level detection. Anti-ICAM-1/catalase quenches hydrogen peroxide levels in the area of injury to physiological levels comparable to that of naive and sham, an effect not achieved with anti-ICAM-1 antibody and catalase alone. Immunohistochemical chomogen staining of 3-NT demonstrates increased tyrosine nitration following CCI-TBI ( D ) compared to naive and sham controls ( B and C , respectively). Anti-ICAM-1/catalase significantly decreased 3-NT detection ( E ). 3-NT levels in anti-ICAM-1 antibody and catalase only groups did not significantly differ from CCI-TBI alone ( F and G , respectively). Positive signal detection was pseudo-colored red for better visualization. All images taken at 20X. Scale bar equals 50 microns. ( H ) Quantification of 3-NT signal detection was analyzed by percent area and normalized to total tissue area per image. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons for hydrogen peroxide data F = 25.95, P

Techniques Used: Immunohistochemistry, Staining, Nitration

In vivo sodium fluorescein assay further elucidates changes in BBB permeability with increased barrier permeability following moderate CCI-TBI and rescue of barrier function with anti-ICAM-1/catalase. Sodium fluorescein was administered IV at 48 hrs post-CCI-TBI to evaluate barrier integrity at this time point. The small molecular weight fluorescent tracer was allowed to circulate for 15 min before tissue perfusion and collection. Fluorescent images captured using short wave UV light demonstrate sodium fluorescein leakage into brain tissue in naive (( A ), no craniectomy) and sham ( B ) mice and mice sustaining moderate CCI-TBI events at 48 hr following injury ( C ) CCI-TBI only, D: CCI-TBI+Anti-ICAM-1/catalase, ( E ) CCI-TBI+anti-ICAM-1 antibody, and F: CCI-TBI+catalase). The dashed circle in B demarcates where the craniectomy was performed. Sodium fluorescein leakage into the brain extends beyond the area of impact following moderate CCI-TBI, highlighted by white arrowheads. Black arrowheads indicate areas of hemorrhage, edema, or swelling of the brain following injury. Coup and contrecoup mechanisms of injury resulting from CCI-TBI can be appreciated by comparing sodium fluorescein signal detection in the area of impact (top of brain section) to the base of the brain directly opposite of the area of impact in CCI-TBI and CCI-TBI+Catalase groups ( C , F ).
Figure Legend Snippet: In vivo sodium fluorescein assay further elucidates changes in BBB permeability with increased barrier permeability following moderate CCI-TBI and rescue of barrier function with anti-ICAM-1/catalase. Sodium fluorescein was administered IV at 48 hrs post-CCI-TBI to evaluate barrier integrity at this time point. The small molecular weight fluorescent tracer was allowed to circulate for 15 min before tissue perfusion and collection. Fluorescent images captured using short wave UV light demonstrate sodium fluorescein leakage into brain tissue in naive (( A ), no craniectomy) and sham ( B ) mice and mice sustaining moderate CCI-TBI events at 48 hr following injury ( C ) CCI-TBI only, D: CCI-TBI+Anti-ICAM-1/catalase, ( E ) CCI-TBI+anti-ICAM-1 antibody, and F: CCI-TBI+catalase). The dashed circle in B demarcates where the craniectomy was performed. Sodium fluorescein leakage into the brain extends beyond the area of impact following moderate CCI-TBI, highlighted by white arrowheads. Black arrowheads indicate areas of hemorrhage, edema, or swelling of the brain following injury. Coup and contrecoup mechanisms of injury resulting from CCI-TBI can be appreciated by comparing sodium fluorescein signal detection in the area of impact (top of brain section) to the base of the brain directly opposite of the area of impact in CCI-TBI and CCI-TBI+Catalase groups ( C , F ).

Techniques Used: In Vivo, Permeability, Molecular Weight, Mouse Assay

CCI-TBI induces local and temporal increase in ICAM-1 expression in mouse cerebral cortex and underlying brain structures. ( A ) Immunohistochemical detection of ICAM-1 time course of expression in cerebral vasculature (solid arrows) and aberrant expression in astrocytes (open arrows) in area of impact in naive (no craniectomy control), sham (surgical control without impact) and at 8, 24, and 48 hr post-CCI-TBI (20X). Included is a stitched coronal section through the CCI-TBI impact site 48 hrs following CCI-TBI (boxed, 4X). Increased ICAM-1 expression detection can also be visualized in hippocampal regions underlying the impact site. ( B ) Quantification of ICAM-1 staining optical density (OD) normalized to vessel caliber and length for naive, sham, and 48 hr post-CCI-TBI groups. Scale bar equals 50 microns. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons F = 96.35, P
Figure Legend Snippet: CCI-TBI induces local and temporal increase in ICAM-1 expression in mouse cerebral cortex and underlying brain structures. ( A ) Immunohistochemical detection of ICAM-1 time course of expression in cerebral vasculature (solid arrows) and aberrant expression in astrocytes (open arrows) in area of impact in naive (no craniectomy control), sham (surgical control without impact) and at 8, 24, and 48 hr post-CCI-TBI (20X). Included is a stitched coronal section through the CCI-TBI impact site 48 hrs following CCI-TBI (boxed, 4X). Increased ICAM-1 expression detection can also be visualized in hippocampal regions underlying the impact site. ( B ) Quantification of ICAM-1 staining optical density (OD) normalized to vessel caliber and length for naive, sham, and 48 hr post-CCI-TBI groups. Scale bar equals 50 microns. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons F = 96.35, P

Techniques Used: Expressing, Immunohistochemistry, Staining

Diminished neuropathology indices with acute anti-ICAM-1/catalase administration following CCI-TBI demonstrated by immunohistochemical staining for neurons, astrocytes, and microglia. ( A ) NeuN staining identifies viable neurons within the cerebral cortex and demonstrates substantial neuronal loss 48 hrs following CCI-TBI. Primary injury mechanisms invariably result in cell death, as indicated by persistently decreased NeuN detection, based on particle count analysis, that is rescued with targeted antioxidant enzyme intervention. NeuN detection following anti-ICAM-1/catalase administration was significantly higher than CCI-TBI alone or in combination with recombinant catalase and anti-ICAM-1. Green arrows point to nuclear stain of NeuN. ( B ) Microglia are finely ramified with small cell bodies in the sham condition. Following CCI-TBI, number of activated microglia per 5.61 × 10 5 microns squared is significantly increased as indicated by threshold increases in cell body size and Iba1 staining intensity. Anti-ICAM-1/catalase reduced the number of amoeboid microglia per frame, while catalase and anti-ICAM-1 alone did not. Red arrows point to amoeboid microglia. ( C ) GFAP expression indicating astrocyte activation shows significant increase at 48 hrs following CCI-TBI compared to optical density readouts per 5.61 × 10 5 microns squared for the sham group. Anti-ICAM-1/catalase significantly reduced optical density of GFAP detection at 48 hrs after CCI-TBI compared to CCI-TBI alone. Non-targeted catalase and anti-ICAM-1 controls did not offer benefit regarding astrocyte activation following CCI-TBI, with no significant difference from CCI-TBI alone. Yellow arrows point to astrocytes with robust GFAP staining detection. Scale bars equal 50 microns. ( D ) Associated bar graphs display imaging quantification. Naive animals were included in image analysis and quantification but are not displayed in the montage. Data are presented as mean ± SD. (Ordinary one-way ANOVA. NeuN: F = 39.83, P
Figure Legend Snippet: Diminished neuropathology indices with acute anti-ICAM-1/catalase administration following CCI-TBI demonstrated by immunohistochemical staining for neurons, astrocytes, and microglia. ( A ) NeuN staining identifies viable neurons within the cerebral cortex and demonstrates substantial neuronal loss 48 hrs following CCI-TBI. Primary injury mechanisms invariably result in cell death, as indicated by persistently decreased NeuN detection, based on particle count analysis, that is rescued with targeted antioxidant enzyme intervention. NeuN detection following anti-ICAM-1/catalase administration was significantly higher than CCI-TBI alone or in combination with recombinant catalase and anti-ICAM-1. Green arrows point to nuclear stain of NeuN. ( B ) Microglia are finely ramified with small cell bodies in the sham condition. Following CCI-TBI, number of activated microglia per 5.61 × 10 5 microns squared is significantly increased as indicated by threshold increases in cell body size and Iba1 staining intensity. Anti-ICAM-1/catalase reduced the number of amoeboid microglia per frame, while catalase and anti-ICAM-1 alone did not. Red arrows point to amoeboid microglia. ( C ) GFAP expression indicating astrocyte activation shows significant increase at 48 hrs following CCI-TBI compared to optical density readouts per 5.61 × 10 5 microns squared for the sham group. Anti-ICAM-1/catalase significantly reduced optical density of GFAP detection at 48 hrs after CCI-TBI compared to CCI-TBI alone. Non-targeted catalase and anti-ICAM-1 controls did not offer benefit regarding astrocyte activation following CCI-TBI, with no significant difference from CCI-TBI alone. Yellow arrows point to astrocytes with robust GFAP staining detection. Scale bars equal 50 microns. ( D ) Associated bar graphs display imaging quantification. Naive animals were included in image analysis and quantification but are not displayed in the montage. Data are presented as mean ± SD. (Ordinary one-way ANOVA. NeuN: F = 39.83, P

Techniques Used: Immunohistochemistry, Staining, Recombinant, Expressing, Activation Assay, Imaging

3) Product Images from "The role for decorin in delayed-type hypersensitivity"

Article Title: The role for decorin in delayed-type hypersensitivity

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1100373

ICAM-1 expression is differentially altered in wild-type and Dcn −/− mice during DTH activity
Figure Legend Snippet: ICAM-1 expression is differentially altered in wild-type and Dcn −/− mice during DTH activity

Techniques Used: Expressing, Mouse Assay, Activity Assay

4) Product Images from "Paracrine Met signaling triggers epithelial–mesenchymal transition in mammary luminal progenitors, affecting their fate"

Article Title: Paracrine Met signaling triggers epithelial–mesenchymal transition in mammary luminal progenitors, affecting their fate

Journal: eLife

doi: 10.7554/eLife.06104

Response of luminal progenitors isolated from Icam1 -deficient mammary epithelium to HGF stimulation. ( A ) Representative images of carmine-stained mammary glands from adult wild-type (WT) and Icam1 -KO mice. V-12w, 12-week-old virgin and P-16d, 16-day pregnant females. Bar, 1.5 mm. ( B ) Flow cytometry analysis of CD24, Itga6, and ICAM-1 expression in mammary epithelial cells freshly isolated from adult virgin WT and Icam1 -KO females. Upper panels: Dot plots of CD24 and Itga6 distribution showing luminal and basal cell compartments. The percentages of luminal cells were equal to 74 ± 9% and 67 ± 9% (data from four independent sorting experiments) in WT and KO epithelium, respectively. Lower panels: Corresponding histograms of ICAM-1 expression in the whole mammary cell population. ( C ) Flow cytometry analysis of CD24, Itga6 and Sca-1 expression in mammary epithelial cells isolated from adult virgin Icam1 -KO females. Left panel: Dot plots of CD24 and Itga6 distribution. Right panel: Dot plots of CD24 and Sca-1 expression in the luminal cell population. ( D ) Representative microphotographs of primary mammospheres derived from purified Icam1 -KO Sca1-neg luminal cells and grown in the presence or absence of HGF for 12 days. ( E ) Characteristics of primary mammospheres derived from purified Icam1 -KO Sca1-neg luminal cells. Left panel: average percentages (±S.E.M) of clonogenic cells in non-stimulated and HGF-stimulated cultures. Data from one cell preparation with three separate wells are shown. Right panel: sphere size distribution (in arbitrary units) in HGF-treated and untreated cultures. At least 250 spheres were analyzed per conditions. ( F ) Krt18 and Krt5 expression levels in spheres derived from untreated and HGF-treated Icam1 -KO Sca1-neg luminal cells. The values were normalized to Gapdh expression. ( G ) Comparative expression levels of basal-specific, EMT-associated and luminal-specific genes in spheres derived from untreated and HGF-treated Icam1 -KO Sca1-neg luminal cells. The q-PCR data are expressed as log 2 ratios of values normalized to Gapdh . The comparator values were those obtained with untreated spheres. DOI: http://dx.doi.org/10.7554/eLife.06104.024
Figure Legend Snippet: Response of luminal progenitors isolated from Icam1 -deficient mammary epithelium to HGF stimulation. ( A ) Representative images of carmine-stained mammary glands from adult wild-type (WT) and Icam1 -KO mice. V-12w, 12-week-old virgin and P-16d, 16-day pregnant females. Bar, 1.5 mm. ( B ) Flow cytometry analysis of CD24, Itga6, and ICAM-1 expression in mammary epithelial cells freshly isolated from adult virgin WT and Icam1 -KO females. Upper panels: Dot plots of CD24 and Itga6 distribution showing luminal and basal cell compartments. The percentages of luminal cells were equal to 74 ± 9% and 67 ± 9% (data from four independent sorting experiments) in WT and KO epithelium, respectively. Lower panels: Corresponding histograms of ICAM-1 expression in the whole mammary cell population. ( C ) Flow cytometry analysis of CD24, Itga6 and Sca-1 expression in mammary epithelial cells isolated from adult virgin Icam1 -KO females. Left panel: Dot plots of CD24 and Itga6 distribution. Right panel: Dot plots of CD24 and Sca-1 expression in the luminal cell population. ( D ) Representative microphotographs of primary mammospheres derived from purified Icam1 -KO Sca1-neg luminal cells and grown in the presence or absence of HGF for 12 days. ( E ) Characteristics of primary mammospheres derived from purified Icam1 -KO Sca1-neg luminal cells. Left panel: average percentages (±S.E.M) of clonogenic cells in non-stimulated and HGF-stimulated cultures. Data from one cell preparation with three separate wells are shown. Right panel: sphere size distribution (in arbitrary units) in HGF-treated and untreated cultures. At least 250 spheres were analyzed per conditions. ( F ) Krt18 and Krt5 expression levels in spheres derived from untreated and HGF-treated Icam1 -KO Sca1-neg luminal cells. The values were normalized to Gapdh expression. ( G ) Comparative expression levels of basal-specific, EMT-associated and luminal-specific genes in spheres derived from untreated and HGF-treated Icam1 -KO Sca1-neg luminal cells. The q-PCR data are expressed as log 2 ratios of values normalized to Gapdh . The comparator values were those obtained with untreated spheres. DOI: http://dx.doi.org/10.7554/eLife.06104.024

Techniques Used: Isolation, Staining, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Derivative Assay, Purification, Polymerase Chain Reaction

Gating procedure for flow cytometry analysis. ( A ) Sequential steps of gating procedure for flow cytometry analysis and sort of mammary epithelial cells stained with anti-CD31, anti-CD45, anti-CD24 and anti-ICAM-1 antibodies. From left to right: exclusion of debris by gating cells on forward (FSC-A) and side scatter (SSC-A) parameters, exclusion of doublets by gating cells on SSC-A and SSC-W parameters, exclusion of CD31/CD45-expressing cells, luminal and basal cell separation using CD24 and ICAM-1 expression. ( B ) Purity control of the sorted ICAM1-neg, ICAM1-low, and ICAM1-hi CD24-positive epithelial cell populations. Cell purity was ≥97%. ( C ) Percentages of ICAM1-neg, ICAM1-low, and ICAM1-hi mammary epithelial cells at puberty, maturity, early-, and late pregnancy. Data are expressed as the mean (±S.E.M) of three flow cytometry analyses. DOI: http://dx.doi.org/10.7554/eLife.06104.005
Figure Legend Snippet: Gating procedure for flow cytometry analysis. ( A ) Sequential steps of gating procedure for flow cytometry analysis and sort of mammary epithelial cells stained with anti-CD31, anti-CD45, anti-CD24 and anti-ICAM-1 antibodies. From left to right: exclusion of debris by gating cells on forward (FSC-A) and side scatter (SSC-A) parameters, exclusion of doublets by gating cells on SSC-A and SSC-W parameters, exclusion of CD31/CD45-expressing cells, luminal and basal cell separation using CD24 and ICAM-1 expression. ( B ) Purity control of the sorted ICAM1-neg, ICAM1-low, and ICAM1-hi CD24-positive epithelial cell populations. Cell purity was ≥97%. ( C ) Percentages of ICAM1-neg, ICAM1-low, and ICAM1-hi mammary epithelial cells at puberty, maturity, early-, and late pregnancy. Data are expressed as the mean (±S.E.M) of three flow cytometry analyses. DOI: http://dx.doi.org/10.7554/eLife.06104.005

Techniques Used: Flow Cytometry, Cytometry, Staining, Expressing

Molecular and phenotypic characteristics of luminal progenitors isolated from virgin and early pregnant females. ( A ) q-PCR analysis of gene expression levels in mammary Lu-neg and Lu-pos cells isolated from early pregnant females (P-8d). Mean ratios of values normalized to Gapdh expression are shown. Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in upper and lower panels, respectively. Data are from two independent cell preparations. ( B ) Icam-1 , Met , and Hgf expression in Lu-neg, Lu-pos, and basal/myoepithelial cells, as determined by q-PCR. Cells were isolated from mammary glands of 8-day-pregnant mice. The q-PCR values were normalized to Gapdh expression and represent mean from two independent preparations. ( C ) Immunodetection of CD44v6 and K8 in cytocentrifuged Lu-neg and Lu-pos cells freshly isolated from mammary glands of mature virgin mice. In upper panels, nuclei were stained with DAPI. Bar, 15 μm. DOI: http://dx.doi.org/10.7554/eLife.06104.009
Figure Legend Snippet: Molecular and phenotypic characteristics of luminal progenitors isolated from virgin and early pregnant females. ( A ) q-PCR analysis of gene expression levels in mammary Lu-neg and Lu-pos cells isolated from early pregnant females (P-8d). Mean ratios of values normalized to Gapdh expression are shown. Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in upper and lower panels, respectively. Data are from two independent cell preparations. ( B ) Icam-1 , Met , and Hgf expression in Lu-neg, Lu-pos, and basal/myoepithelial cells, as determined by q-PCR. Cells were isolated from mammary glands of 8-day-pregnant mice. The q-PCR values were normalized to Gapdh expression and represent mean from two independent preparations. ( C ) Immunodetection of CD44v6 and K8 in cytocentrifuged Lu-neg and Lu-pos cells freshly isolated from mammary glands of mature virgin mice. In upper panels, nuclei were stained with DAPI. Bar, 15 μm. DOI: http://dx.doi.org/10.7554/eLife.06104.009

Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, Mouse Assay, Immunodetection, Staining

Met expression in luminal progenitors isolated from Blg-Cre; R26 mice. ( A ) Icam-1 , Met , and Hgf expression in Lu-neg, Lu-pos, and basal/myoepithelial cells, as determined by q-PCR. Cells were isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. The q-PCR values were normalized to Gapdh expression. ( B ) Percentages of K5-positive cells (upper panel) and K5-positive cells expressing LacZ (lower panel) in HGF-treated spheres derived from Lu-pos and basal cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. Data are from one cell preparation. At least 500 cells were counted per sample. DOI: http://dx.doi.org/10.7554/eLife.06104.015
Figure Legend Snippet: Met expression in luminal progenitors isolated from Blg-Cre; R26 mice. ( A ) Icam-1 , Met , and Hgf expression in Lu-neg, Lu-pos, and basal/myoepithelial cells, as determined by q-PCR. Cells were isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. The q-PCR values were normalized to Gapdh expression. ( B ) Percentages of K5-positive cells (upper panel) and K5-positive cells expressing LacZ (lower panel) in HGF-treated spheres derived from Lu-pos and basal cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. Data are from one cell preparation. At least 500 cells were counted per sample. DOI: http://dx.doi.org/10.7554/eLife.06104.015

Techniques Used: Expressing, Isolation, Mouse Assay, Polymerase Chain Reaction, Derivative Assay

Characterization of luminal cell populations isolated from virgin and pregnant mice using ICAM-1 and Sca-1 expression. ( A ) Flow cytometry dot plots showing the gating procedure of the four luminal cell subsets (Lu1, Lu2, Lu3, and Lu4) identified by Sca-1 and ICAM-1. Cells were isolated from mammary glands of mature virgin mice. ( B ) qPCR analysis of Krt18 and Krt14 expression in Lu1, Lu2, Lu3, and Lu4 cells isolated from mammary glands of mature virgin mice as shown in ( A ). Mean values ±S.E.M. from three independent cell preparations are shown. ( C ) Percentages of clonogenic cells in Lu1, Lu2, Lu3, and Lu4 populations isolated from mammary glands of mice taken at different stages of development: V-6w, 6-week-old virgin; P-8d, 8-day pregnant; P-16d, 16-day pregnant mice. Corresponding flow cytometry dot plots are shown in Figure 8A . Two separate cell preparations (distributed in three wells) were analyzed at each stages of development. The results are presented as mean values ±S.E.M. (*) No cell population. ( D ) Heat map of qPCR gene expression analysis performed on Lu1, Lu2, Lu3, and Lu4 cells freshly isolated from mammary glands of 8-day pregnant mice (P8-d). The qPCR values were normalized to Gapdh expression. Mean values from two independent cell preparations were used to establish the map and determine relationships between the luminal subsets by unsupervised hierarchical clustering. ( E ) q-PCR analysis of Icam-1 and Met expression in Lu1, Lu2, Lu3, and Lu4 cell populations freshly isolated from mammary glands of 8-day pregnant mice (P8-d). The values were normalized to Gapdh expression and represent means from two independent preparations. DOI: http://dx.doi.org/10.7554/eLife.06104.022
Figure Legend Snippet: Characterization of luminal cell populations isolated from virgin and pregnant mice using ICAM-1 and Sca-1 expression. ( A ) Flow cytometry dot plots showing the gating procedure of the four luminal cell subsets (Lu1, Lu2, Lu3, and Lu4) identified by Sca-1 and ICAM-1. Cells were isolated from mammary glands of mature virgin mice. ( B ) qPCR analysis of Krt18 and Krt14 expression in Lu1, Lu2, Lu3, and Lu4 cells isolated from mammary glands of mature virgin mice as shown in ( A ). Mean values ±S.E.M. from three independent cell preparations are shown. ( C ) Percentages of clonogenic cells in Lu1, Lu2, Lu3, and Lu4 populations isolated from mammary glands of mice taken at different stages of development: V-6w, 6-week-old virgin; P-8d, 8-day pregnant; P-16d, 16-day pregnant mice. Corresponding flow cytometry dot plots are shown in Figure 8A . Two separate cell preparations (distributed in three wells) were analyzed at each stages of development. The results are presented as mean values ±S.E.M. (*) No cell population. ( D ) Heat map of qPCR gene expression analysis performed on Lu1, Lu2, Lu3, and Lu4 cells freshly isolated from mammary glands of 8-day pregnant mice (P8-d). The qPCR values were normalized to Gapdh expression. Mean values from two independent cell preparations were used to establish the map and determine relationships between the luminal subsets by unsupervised hierarchical clustering. ( E ) q-PCR analysis of Icam-1 and Met expression in Lu1, Lu2, Lu3, and Lu4 cell populations freshly isolated from mammary glands of 8-day pregnant mice (P8-d). The values were normalized to Gapdh expression and represent means from two independent preparations. DOI: http://dx.doi.org/10.7554/eLife.06104.022

Techniques Used: Isolation, Mouse Assay, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Isolation of mammary luminal progenitors from adult virgin C57Bl/6J and Blg-Cre; R26 females using ICAM-1. ( A ) Isolation of clonogenic luminal progenitors from adult virgin C57Bl/6J mice using ICAM-1. Left panel: flow cytometry analysis of ICAM-1 and CD24 expression in freshly isolated mammary epithelial cells. Middle panel: H E staining of clonal colonies obtained from Lu-neg and Lu-pos luminal cells after 8 days in culture. Right panel: percentages of clonogenic cells. The results are from triplicates obtained with one cell preparation and presented as mean values ±S.E.M. ( B ) Flow cytometry analysis of ICAM-1 and CD24 expression in mammary epithelial cells freshly isolated from adult virgin Blg-Cre; R26 females. ( C ) Sections through Blg-Cre; R26 mouse mammary gland Xgal-stained in whole mount. Blue and white arrows indicate LacZ-positive luminal cells and LacZ-negative basal cells, respectively. Bar, 15 μm. ( D ) Icam-1 and Cre expression in Lu-neg, Lu-pos, and basal cells, as determined by q-PCR. The values normalized to Gapdh expression are from one representative experiment performed with 3 pooled adult virgin Blg-Cre; R26 mice. ( E ) Clonogenic potential Lu-neg and Lu-pos luminal cells isolated from adult virgin Blg-Cre; R26 mice using ICAM-1. Left panel: Xgal staining of colonies counterstained with fast red. Right panel: percentages of clonogenic cells. The results are from triplicates obtained with one cell preparation and presented as mean values ±S.E.M. ( F ) q-PCR analysis of gene expression levels in Lu-neg and Lu-pos cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. Ratios of values normalized to Gapdh expression are shown. Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in left and right panels, respectively. DOI: http://dx.doi.org/10.7554/eLife.06104.006
Figure Legend Snippet: Isolation of mammary luminal progenitors from adult virgin C57Bl/6J and Blg-Cre; R26 females using ICAM-1. ( A ) Isolation of clonogenic luminal progenitors from adult virgin C57Bl/6J mice using ICAM-1. Left panel: flow cytometry analysis of ICAM-1 and CD24 expression in freshly isolated mammary epithelial cells. Middle panel: H E staining of clonal colonies obtained from Lu-neg and Lu-pos luminal cells after 8 days in culture. Right panel: percentages of clonogenic cells. The results are from triplicates obtained with one cell preparation and presented as mean values ±S.E.M. ( B ) Flow cytometry analysis of ICAM-1 and CD24 expression in mammary epithelial cells freshly isolated from adult virgin Blg-Cre; R26 females. ( C ) Sections through Blg-Cre; R26 mouse mammary gland Xgal-stained in whole mount. Blue and white arrows indicate LacZ-positive luminal cells and LacZ-negative basal cells, respectively. Bar, 15 μm. ( D ) Icam-1 and Cre expression in Lu-neg, Lu-pos, and basal cells, as determined by q-PCR. The values normalized to Gapdh expression are from one representative experiment performed with 3 pooled adult virgin Blg-Cre; R26 mice. ( E ) Clonogenic potential Lu-neg and Lu-pos luminal cells isolated from adult virgin Blg-Cre; R26 mice using ICAM-1. Left panel: Xgal staining of colonies counterstained with fast red. Right panel: percentages of clonogenic cells. The results are from triplicates obtained with one cell preparation and presented as mean values ±S.E.M. ( F ) q-PCR analysis of gene expression levels in Lu-neg and Lu-pos cells isolated from mammary glands of 3 pooled adult virgin Blg-Cre; R26 mice. Ratios of values normalized to Gapdh expression are shown. Lu-neg/Lu-pos and Lu-pos/Lu-neg ratios are presented in left and right panels, respectively. DOI: http://dx.doi.org/10.7554/eLife.06104.006

Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, Polymerase Chain Reaction

5) Product Images from "Platelet expression of tumour necrosis factor-alpha (TNF-?), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy"

Article Title: Platelet expression of tumour necrosis factor-alpha (TNF-?), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.1999.01067.x

Relationship between the expression of TNF-α and that of TNF-RI, TNF-RII and ICAM-1 in platelets from individuals with insulin-dependent diabetes mellitus (IDDM) complicated by proliferative diabetic retinopathy (PDR).
Figure Legend Snippet: Relationship between the expression of TNF-α and that of TNF-RI, TNF-RII and ICAM-1 in platelets from individuals with insulin-dependent diabetes mellitus (IDDM) complicated by proliferative diabetic retinopathy (PDR).

Techniques Used: Expressing

Percentage of platelets staining for ICAM-1 in patients with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR), quiescent PDR and healthy subjects. Mann–Whitney U -tests: * P = 0.0065 ( versus quiescent PDR), P = 0.013 ( versus healthy subjects); ** P = 0.036 ( versus quiescent PDR); P = 0.047 ( versus healthy subjects). The dotted line represents the median of the number of ICAM-1-positive platelets in healthy subjects.
Figure Legend Snippet: Percentage of platelets staining for ICAM-1 in patients with insulin-dependent diabetes mellitus (IDDM) complicated or uncomplicated by proliferative diabetic retinopathy (PDR), quiescent PDR and healthy subjects. Mann–Whitney U -tests: * P = 0.0065 ( versus quiescent PDR), P = 0.013 ( versus healthy subjects); ** P = 0.036 ( versus quiescent PDR); P = 0.047 ( versus healthy subjects). The dotted line represents the median of the number of ICAM-1-positive platelets in healthy subjects.

Techniques Used: Staining, MANN-WHITNEY

6) Product Images from "The Impact of Ischemia/Reperfusion Injury on Liver Allografts from Deceased after Cardiac Death versus Deceased after Brain Death Donors"

Article Title: The Impact of Ischemia/Reperfusion Injury on Liver Allografts from Deceased after Cardiac Death versus Deceased after Brain Death Donors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0148815

Immunohistochemistry results prior to transplantation. (A) Leukocyte infiltration and FasL expression in liver allografts prior to transplantation. Biopsies from DBD and DCD liver biopsies were stained with monoclonal antibodies against neutrophil elastase, CD3 and FasL. (B) Expression of ICAM-1 in liver allografts prior to transplantation.
Figure Legend Snippet: Immunohistochemistry results prior to transplantation. (A) Leukocyte infiltration and FasL expression in liver allografts prior to transplantation. Biopsies from DBD and DCD liver biopsies were stained with monoclonal antibodies against neutrophil elastase, CD3 and FasL. (B) Expression of ICAM-1 in liver allografts prior to transplantation.

Techniques Used: Immunohistochemistry, Transplantation Assay, Expressing, Staining

7) Product Images from "The Junctional Adhesion Molecule 3 (JAM-3) on Human Platelets is a Counterreceptor for the Leukocyte Integrin Mac-1"

Article Title: The Junctional Adhesion Molecule 3 (JAM-3) on Human Platelets is a Counterreceptor for the Leukocyte Integrin Mac-1

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20020267

Interaction between purified JAM-3 and Mac-1 proteins. (A) The binding of ICAM-1, fibrinogen (FBG), or JAM-3 to immobilized Mac-1 (black bars) and the binding of JAM-3 or ICAM-1 to immobilized LFA-1 (white bars) was studied. (B) Dose-dependent specific binding of JAM-3 to immobilized Mac-1 is shown. (C) The binding of JAM-3 (white bars) to immobilized Mac-1 was analyzed in the absence (−) or presence of fibrinogen (FBG, 20 μg/ml), mAb against Mac-1 or mAb against JAM-3 (each 10 μg/ml). The binding of fibrinogen (black bars) to immobilized Mac-1 was studied in the absence (−) or presence of purified JAM-3 (20 μg/ml) or of mAb against Mac-1 (10 μg/ml). Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD ( n = 3) of a typical experiment; similar results were observed in at least three separate experiments.
Figure Legend Snippet: Interaction between purified JAM-3 and Mac-1 proteins. (A) The binding of ICAM-1, fibrinogen (FBG), or JAM-3 to immobilized Mac-1 (black bars) and the binding of JAM-3 or ICAM-1 to immobilized LFA-1 (white bars) was studied. (B) Dose-dependent specific binding of JAM-3 to immobilized Mac-1 is shown. (C) The binding of JAM-3 (white bars) to immobilized Mac-1 was analyzed in the absence (−) or presence of fibrinogen (FBG, 20 μg/ml), mAb against Mac-1 or mAb against JAM-3 (each 10 μg/ml). The binding of fibrinogen (black bars) to immobilized Mac-1 was studied in the absence (−) or presence of purified JAM-3 (20 μg/ml) or of mAb against Mac-1 (10 μg/ml). Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD ( n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Techniques Used: Purification, Binding Assay

8) Product Images from "Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury"

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury

Journal: Scientific Reports

doi: 10.1038/s41598-017-03309-4

CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).
Figure Legend Snippet: CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).

Techniques Used: Permeability, Preserving, Immunohistochemistry, Staining, Western Blot, Expressing

Two-photon imaging detection of microglia in CX3CR1-GFP mice and morphometric analysis of cellular morphology in CCI-TBI and anti-ICAM-1/catalase administration. CX3CR1-GFP mice (B6.129P- Cx3cr1 tm1Litt /J, The Jackson Laboratory) were subjected to moderate CCI-TBI with and without anti-ICAM-1/catalase conjugate administration 30 min after impact. No craniectomy (naive) CX3CR1-GFP mice served as control. At 48 hrs following CCI-TBI, subjects were perfusion fixed, brain tissue was collected and subsequently sectioned into 1 mm segments for two-photon imaging. ( A ) Representative single slice images depict microglial ramification in the area of impact for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase mice. Arrowheads point to microglial processes, which are finely ramified in the naive condition and thicken and retract following CCI-TBI. Anti-ICAM-1/catalase attenuates changes in microglia cell body enlargement and process retraction. Scale bar 20 μm. Imaris (Bitplane) software was utilized for 3D reconstruction of GFP-expressing microglia from z-stack images obtained by two-photon microscopy. ( B ) Surface rendering was performed for microglia based on GFP signal threshold to measure cellular surface area ( D ) (only objects with surface area > 1000 μm 2 were analyzed) and sphericity ( E ). ( C ) Imaris FilamentTracer was employed to map microglial processes to quantify changes in cell ramification in CCI-TBI. Microglial total filament length ( F ) and number of filament branching points (complexity, ( G ) and were assessed for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase groups. Data presented at mean ± SD. (Ordinary one-way ANOVA. Surface area: F = 73.12, P
Figure Legend Snippet: Two-photon imaging detection of microglia in CX3CR1-GFP mice and morphometric analysis of cellular morphology in CCI-TBI and anti-ICAM-1/catalase administration. CX3CR1-GFP mice (B6.129P- Cx3cr1 tm1Litt /J, The Jackson Laboratory) were subjected to moderate CCI-TBI with and without anti-ICAM-1/catalase conjugate administration 30 min after impact. No craniectomy (naive) CX3CR1-GFP mice served as control. At 48 hrs following CCI-TBI, subjects were perfusion fixed, brain tissue was collected and subsequently sectioned into 1 mm segments for two-photon imaging. ( A ) Representative single slice images depict microglial ramification in the area of impact for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase mice. Arrowheads point to microglial processes, which are finely ramified in the naive condition and thicken and retract following CCI-TBI. Anti-ICAM-1/catalase attenuates changes in microglia cell body enlargement and process retraction. Scale bar 20 μm. Imaris (Bitplane) software was utilized for 3D reconstruction of GFP-expressing microglia from z-stack images obtained by two-photon microscopy. ( B ) Surface rendering was performed for microglia based on GFP signal threshold to measure cellular surface area ( D ) (only objects with surface area > 1000 μm 2 were analyzed) and sphericity ( E ). ( C ) Imaris FilamentTracer was employed to map microglial processes to quantify changes in cell ramification in CCI-TBI. Microglial total filament length ( F ) and number of filament branching points (complexity, ( G ) and were assessed for naive, CCI-TBI, and CCI-TBI+anti-ICAM-1/catalase groups. Data presented at mean ± SD. (Ordinary one-way ANOVA. Surface area: F = 73.12, P

Techniques Used: Imaging, Mouse Assay, Software, Expressing, Microscopy

CCI-TBI results in increased hydrogen peroxide production and 3-nitrotyrosine detection in the ipsilateral cortex area of impact. ( A ) OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit was used to measure hydrogen peroxide levels in fresh mouse cortical tissue ipsilateral to the area of impact 4 hrs following CCI-TBI, CCI-TBI+anti-ICAM-1/catalase, catalase only, and antibody only administration or from naive and sham controls. The naive condition provides basal hydrogen peroxide levels in uninjured cortical tissue which are not significantly different from sham. CCI-TBI results in a nearly 3-fold increase in hydrogen peroxide level detection. Anti-ICAM-1/catalase quenches hydrogen peroxide levels in the area of injury to physiological levels comparable to that of naive and sham, an effect not achieved with anti-ICAM-1 antibody and catalase alone. Immunohistochemical chomogen staining of 3-NT demonstrates increased tyrosine nitration following CCI-TBI ( D ) compared to naive and sham controls ( B and C , respectively). Anti-ICAM-1/catalase significantly decreased 3-NT detection ( E ). 3-NT levels in anti-ICAM-1 antibody and catalase only groups did not significantly differ from CCI-TBI alone ( F and G , respectively). Positive signal detection was pseudo-colored red for better visualization. All images taken at 20X. Scale bar equals 50 microns. ( H ) Quantification of 3-NT signal detection was analyzed by percent area and normalized to total tissue area per image. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons for hydrogen peroxide data F = 25.95, P
Figure Legend Snippet: CCI-TBI results in increased hydrogen peroxide production and 3-nitrotyrosine detection in the ipsilateral cortex area of impact. ( A ) OxiSelect™ Hydrogen Peroxide/Peroxidase Assay Kit was used to measure hydrogen peroxide levels in fresh mouse cortical tissue ipsilateral to the area of impact 4 hrs following CCI-TBI, CCI-TBI+anti-ICAM-1/catalase, catalase only, and antibody only administration or from naive and sham controls. The naive condition provides basal hydrogen peroxide levels in uninjured cortical tissue which are not significantly different from sham. CCI-TBI results in a nearly 3-fold increase in hydrogen peroxide level detection. Anti-ICAM-1/catalase quenches hydrogen peroxide levels in the area of injury to physiological levels comparable to that of naive and sham, an effect not achieved with anti-ICAM-1 antibody and catalase alone. Immunohistochemical chomogen staining of 3-NT demonstrates increased tyrosine nitration following CCI-TBI ( D ) compared to naive and sham controls ( B and C , respectively). Anti-ICAM-1/catalase significantly decreased 3-NT detection ( E ). 3-NT levels in anti-ICAM-1 antibody and catalase only groups did not significantly differ from CCI-TBI alone ( F and G , respectively). Positive signal detection was pseudo-colored red for better visualization. All images taken at 20X. Scale bar equals 50 microns. ( H ) Quantification of 3-NT signal detection was analyzed by percent area and normalized to total tissue area per image. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons for hydrogen peroxide data F = 25.95, P

Techniques Used: Immunohistochemistry, Staining, Nitration

In vivo sodium fluorescein assay further elucidates changes in BBB permeability with increased barrier permeability following moderate CCI-TBI and rescue of barrier function with anti-ICAM-1/catalase. Sodium fluorescein was administered IV at 48 hrs post-CCI-TBI to evaluate barrier integrity at this time point. The small molecular weight fluorescent tracer was allowed to circulate for 15 min before tissue perfusion and collection. Fluorescent images captured using short wave UV light demonstrate sodium fluorescein leakage into brain tissue in naive (( A ), no craniectomy) and sham ( B ) mice and mice sustaining moderate CCI-TBI events at 48 hr following injury ( C ) CCI-TBI only, D: CCI-TBI+Anti-ICAM-1/catalase, ( E ) CCI-TBI+anti-ICAM-1 antibody, and F: CCI-TBI+catalase). The dashed circle in B demarcates where the craniectomy was performed. Sodium fluorescein leakage into the brain extends beyond the area of impact following moderate CCI-TBI, highlighted by white arrowheads. Black arrowheads indicate areas of hemorrhage, edema, or swelling of the brain following injury. Coup and contrecoup mechanisms of injury resulting from CCI-TBI can be appreciated by comparing sodium fluorescein signal detection in the area of impact (top of brain section) to the base of the brain directly opposite of the area of impact in CCI-TBI and CCI-TBI+Catalase groups ( C , F ).
Figure Legend Snippet: In vivo sodium fluorescein assay further elucidates changes in BBB permeability with increased barrier permeability following moderate CCI-TBI and rescue of barrier function with anti-ICAM-1/catalase. Sodium fluorescein was administered IV at 48 hrs post-CCI-TBI to evaluate barrier integrity at this time point. The small molecular weight fluorescent tracer was allowed to circulate for 15 min before tissue perfusion and collection. Fluorescent images captured using short wave UV light demonstrate sodium fluorescein leakage into brain tissue in naive (( A ), no craniectomy) and sham ( B ) mice and mice sustaining moderate CCI-TBI events at 48 hr following injury ( C ) CCI-TBI only, D: CCI-TBI+Anti-ICAM-1/catalase, ( E ) CCI-TBI+anti-ICAM-1 antibody, and F: CCI-TBI+catalase). The dashed circle in B demarcates where the craniectomy was performed. Sodium fluorescein leakage into the brain extends beyond the area of impact following moderate CCI-TBI, highlighted by white arrowheads. Black arrowheads indicate areas of hemorrhage, edema, or swelling of the brain following injury. Coup and contrecoup mechanisms of injury resulting from CCI-TBI can be appreciated by comparing sodium fluorescein signal detection in the area of impact (top of brain section) to the base of the brain directly opposite of the area of impact in CCI-TBI and CCI-TBI+Catalase groups ( C , F ).

Techniques Used: In Vivo, Permeability, Molecular Weight, Mouse Assay

CCI-TBI induces local and temporal increase in ICAM-1 expression in mouse cerebral cortex and underlying brain structures. ( A ) Immunohistochemical detection of ICAM-1 time course of expression in cerebral vasculature (solid arrows) and aberrant expression in astrocytes (open arrows) in area of impact in naive (no craniectomy control), sham (surgical control without impact) and at 8, 24, and 48 hr post-CCI-TBI (20X). Included is a stitched coronal section through the CCI-TBI impact site 48 hrs following CCI-TBI (boxed, 4X). Increased ICAM-1 expression detection can also be visualized in hippocampal regions underlying the impact site. ( B ) Quantification of ICAM-1 staining optical density (OD) normalized to vessel caliber and length for naive, sham, and 48 hr post-CCI-TBI groups. Scale bar equals 50 microns. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons F = 96.35, P
Figure Legend Snippet: CCI-TBI induces local and temporal increase in ICAM-1 expression in mouse cerebral cortex and underlying brain structures. ( A ) Immunohistochemical detection of ICAM-1 time course of expression in cerebral vasculature (solid arrows) and aberrant expression in astrocytes (open arrows) in area of impact in naive (no craniectomy control), sham (surgical control without impact) and at 8, 24, and 48 hr post-CCI-TBI (20X). Included is a stitched coronal section through the CCI-TBI impact site 48 hrs following CCI-TBI (boxed, 4X). Increased ICAM-1 expression detection can also be visualized in hippocampal regions underlying the impact site. ( B ) Quantification of ICAM-1 staining optical density (OD) normalized to vessel caliber and length for naive, sham, and 48 hr post-CCI-TBI groups. Scale bar equals 50 microns. Data are presented as mean ± SD. (Ordinary one-way ANOVA with multiple comparisons F = 96.35, P

Techniques Used: Expressing, Immunohistochemistry, Staining

Diminished neuropathology indices with acute anti-ICAM-1/catalase administration following CCI-TBI demonstrated by immunohistochemical staining for neurons, astrocytes, and microglia. ( A ) NeuN staining identifies viable neurons within the cerebral cortex and demonstrates substantial neuronal loss 48 hrs following CCI-TBI. Primary injury mechanisms invariably result in cell death, as indicated by persistently decreased NeuN detection, based on particle count analysis, that is rescued with targeted antioxidant enzyme intervention. NeuN detection following anti-ICAM-1/catalase administration was significantly higher than CCI-TBI alone or in combination with recombinant catalase and anti-ICAM-1. Green arrows point to nuclear stain of NeuN. ( B ) Microglia are finely ramified with small cell bodies in the sham condition. Following CCI-TBI, number of activated microglia per 5.61 × 10 5 microns squared is significantly increased as indicated by threshold increases in cell body size and Iba1 staining intensity. Anti-ICAM-1/catalase reduced the number of amoeboid microglia per frame, while catalase and anti-ICAM-1 alone did not. Red arrows point to amoeboid microglia. ( C ) GFAP expression indicating astrocyte activation shows significant increase at 48 hrs following CCI-TBI compared to optical density readouts per 5.61 × 10 5 microns squared for the sham group. Anti-ICAM-1/catalase significantly reduced optical density of GFAP detection at 48 hrs after CCI-TBI compared to CCI-TBI alone. Non-targeted catalase and anti-ICAM-1 controls did not offer benefit regarding astrocyte activation following CCI-TBI, with no significant difference from CCI-TBI alone. Yellow arrows point to astrocytes with robust GFAP staining detection. Scale bars equal 50 microns. ( D ) Associated bar graphs display imaging quantification. Naive animals were included in image analysis and quantification but are not displayed in the montage. Data are presented as mean ± SD. (Ordinary one-way ANOVA. NeuN: F = 39.83, P
Figure Legend Snippet: Diminished neuropathology indices with acute anti-ICAM-1/catalase administration following CCI-TBI demonstrated by immunohistochemical staining for neurons, astrocytes, and microglia. ( A ) NeuN staining identifies viable neurons within the cerebral cortex and demonstrates substantial neuronal loss 48 hrs following CCI-TBI. Primary injury mechanisms invariably result in cell death, as indicated by persistently decreased NeuN detection, based on particle count analysis, that is rescued with targeted antioxidant enzyme intervention. NeuN detection following anti-ICAM-1/catalase administration was significantly higher than CCI-TBI alone or in combination with recombinant catalase and anti-ICAM-1. Green arrows point to nuclear stain of NeuN. ( B ) Microglia are finely ramified with small cell bodies in the sham condition. Following CCI-TBI, number of activated microglia per 5.61 × 10 5 microns squared is significantly increased as indicated by threshold increases in cell body size and Iba1 staining intensity. Anti-ICAM-1/catalase reduced the number of amoeboid microglia per frame, while catalase and anti-ICAM-1 alone did not. Red arrows point to amoeboid microglia. ( C ) GFAP expression indicating astrocyte activation shows significant increase at 48 hrs following CCI-TBI compared to optical density readouts per 5.61 × 10 5 microns squared for the sham group. Anti-ICAM-1/catalase significantly reduced optical density of GFAP detection at 48 hrs after CCI-TBI compared to CCI-TBI alone. Non-targeted catalase and anti-ICAM-1 controls did not offer benefit regarding astrocyte activation following CCI-TBI, with no significant difference from CCI-TBI alone. Yellow arrows point to astrocytes with robust GFAP staining detection. Scale bars equal 50 microns. ( D ) Associated bar graphs display imaging quantification. Naive animals were included in image analysis and quantification but are not displayed in the montage. Data are presented as mean ± SD. (Ordinary one-way ANOVA. NeuN: F = 39.83, P

Techniques Used: Immunohistochemistry, Staining, Recombinant, Expressing, Activation Assay, Imaging

9) Product Images from "Characterization of the inflammatory cells in ascending thoracic aortic aneurysms in patients with Marfan syndrome, familial thoracic aortic aneurysms and sporadic aneurysms"

Article Title: Characterization of the inflammatory cells in ascending thoracic aortic aneurysms in patients with Marfan syndrome, familial thoracic aortic aneurysms and sporadic aneurysms

Journal:

doi: 10.1016/j.jtcvs.2007.12.063

Increased expression of leukocyte adhesion molecules, ICAM-1 and VCAM-1, in the vasa vasorum in the adventitia of MFS and FTAA patients. Immunohistochemistry revealed positive immunostaining for ICAM-1 and VCAM-1 in the vasa vasorum from patients with
Figure Legend Snippet: Increased expression of leukocyte adhesion molecules, ICAM-1 and VCAM-1, in the vasa vasorum in the adventitia of MFS and FTAA patients. Immunohistochemistry revealed positive immunostaining for ICAM-1 and VCAM-1 in the vasa vasorum from patients with

Techniques Used: Expressing, Immunohistochemistry, Immunostaining

10) Product Images from "Effect of Ciprofloxacin-Induced Prostaglandin E2 on Interleukin-18-Treated Monocytes"

Article Title: Effect of Ciprofloxacin-Induced Prostaglandin E2 on Interleukin-18-Treated Monocytes

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.49.8.3228-3233.2005

The effect of CIP on the expression of ICAM-1, B7.1, B7.2, and CD40 on monocytes. (A) Monocytes at 10 6 cells/ml were incubated with between 0 and 100 μg/ml CIP in the presence and absence of 100 ng/ml IL-18 for 24 h. After treatment, the expression of ICAM-1, B7.1, B7.2, and CD40 was determined by flow cytometry. Filled circles and filled squares represent the results obtained with medium and IL-18, respectively. Open circles and open squares represent the class-matched control (IgG1) in the presence and absence of IL-18. *, P
Figure Legend Snippet: The effect of CIP on the expression of ICAM-1, B7.1, B7.2, and CD40 on monocytes. (A) Monocytes at 10 6 cells/ml were incubated with between 0 and 100 μg/ml CIP in the presence and absence of 100 ng/ml IL-18 for 24 h. After treatment, the expression of ICAM-1, B7.1, B7.2, and CD40 was determined by flow cytometry. Filled circles and filled squares represent the results obtained with medium and IL-18, respectively. Open circles and open squares represent the class-matched control (IgG1) in the presence and absence of IL-18. *, P

Techniques Used: Expressing, Incubation, Flow Cytometry, Cytometry

11) Product Images from "Pulmonary exposure to carbon black nanoparticles and vascular effects"

Article Title: Pulmonary exposure to carbon black nanoparticles and vascular effects

Journal: Particle and Fibre Toxicology

doi: 10.1186/1743-8977-7-33

Expression of VCAM-1 and ICAM-1 on the endothelium and the presence of 3-nitrotyrosine (3NT) in the vascular tissue of aorta from 11-13 weeks old apoE -/- mice exposed to carbon black by i.t. instillation . The antibodies used were HRP-streptavidin conjugated and visualized with DAB. On the left, the data are depicted as geometric means of ranked values representing the amount of staining assessed in 4 sections of aorta from each animal. The medians are depicted as horizontal lines (n = 10-20); each symbol represent the result from one animal. On the right, images are shown of aorta sections representative of the median in the control group (left) and the 2.7 mg/kg exposed group (right). Binding of antibodies can be seen as brown staining of the tissue.
Figure Legend Snippet: Expression of VCAM-1 and ICAM-1 on the endothelium and the presence of 3-nitrotyrosine (3NT) in the vascular tissue of aorta from 11-13 weeks old apoE -/- mice exposed to carbon black by i.t. instillation . The antibodies used were HRP-streptavidin conjugated and visualized with DAB. On the left, the data are depicted as geometric means of ranked values representing the amount of staining assessed in 4 sections of aorta from each animal. The medians are depicted as horizontal lines (n = 10-20); each symbol represent the result from one animal. On the right, images are shown of aorta sections representative of the median in the control group (left) and the 2.7 mg/kg exposed group (right). Binding of antibodies can be seen as brown staining of the tissue.

Techniques Used: Expressing, Mouse Assay, Staining, Binding Assay

Expression of VCAM-1 and ICAM-1 on the endothelium and the presence of 3-nitrotyrosine (3NT) in the vascular tissue of brachiocephalic arteries (BCA) from 48-49 weeks old apoE -/- mice exposed to carbon black by i.t. instillation . The antibodies used were HRP-streptavidin conjugated and visualized with DAB. On the left, the data are depicted as geometric means of ranked values representing the amount of staining assessed in 4 sections of aorta from each animal. The medians are represented by horizontal lines (n = 9-11); each symbol represent the result from one animal. On the right, images are shown of BCA sections representative of the medians in the control group (left) and the exposed group (right). Binding of antibodies can be seen as brown staining of the tissue.
Figure Legend Snippet: Expression of VCAM-1 and ICAM-1 on the endothelium and the presence of 3-nitrotyrosine (3NT) in the vascular tissue of brachiocephalic arteries (BCA) from 48-49 weeks old apoE -/- mice exposed to carbon black by i.t. instillation . The antibodies used were HRP-streptavidin conjugated and visualized with DAB. On the left, the data are depicted as geometric means of ranked values representing the amount of staining assessed in 4 sections of aorta from each animal. The medians are represented by horizontal lines (n = 9-11); each symbol represent the result from one animal. On the right, images are shown of BCA sections representative of the medians in the control group (left) and the exposed group (right). Binding of antibodies can be seen as brown staining of the tissue.

Techniques Used: Expressing, BIA-KA, Mouse Assay, Staining, Binding Assay

12) Product Images from "Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts"

Article Title: Leoligin, the major lignan from Edelweiss, inhibits intimal hyperplasia of venous bypass grafts

Journal: Cardiovascular Research

doi: 10.1093/cvr/cvp059

Leoligin is non-toxic for ECs and inhibits TNF-α-mediated VCAM-1 expression. Primary human vascular endothelial cells (ECs) were incubated with the indicated concentrations of leoligin for the indicated times. The upper left diagram shows a representative analysis of cell viability determined by the annexin V/propidium iodide staining and FACS analyses. Values are means of a representative experiment performed in triplicate. The upper right diagram shows EC proliferation as determined by the XTT assay. Values are means of three independent experiments ± SD. The impact of leoligin on TNF-α-induced surface expression of VCAM-1, ICAM-1, and E-selectin (E-Sel) is shown in the lower left diagram. Data shown are mean fluorescence intensities (MFI), % of control, of a representative experiment. The experiment was repeated three times. The lower right image shows a metabolic protein labelling of ECs in the presence of the indicated concentrations of leoligin. The experiment was repeated three times, giving similar results. A representative blot is shown.
Figure Legend Snippet: Leoligin is non-toxic for ECs and inhibits TNF-α-mediated VCAM-1 expression. Primary human vascular endothelial cells (ECs) were incubated with the indicated concentrations of leoligin for the indicated times. The upper left diagram shows a representative analysis of cell viability determined by the annexin V/propidium iodide staining and FACS analyses. Values are means of a representative experiment performed in triplicate. The upper right diagram shows EC proliferation as determined by the XTT assay. Values are means of three independent experiments ± SD. The impact of leoligin on TNF-α-induced surface expression of VCAM-1, ICAM-1, and E-selectin (E-Sel) is shown in the lower left diagram. Data shown are mean fluorescence intensities (MFI), % of control, of a representative experiment. The experiment was repeated three times. The lower right image shows a metabolic protein labelling of ECs in the presence of the indicated concentrations of leoligin. The experiment was repeated three times, giving similar results. A representative blot is shown.

Techniques Used: Expressing, Incubation, Staining, FACS, XTT Assay, Fluorescence

Related Articles

Immunohistochemistry:

Article Title: Paracrine Met signaling triggers epithelial–mesenchymal transition in mammary luminal progenitors, affecting their fate
Article Snippet: .. For the immunohistochemical detection of ICAM-1, we used the EnVision System from Dako. .. Image acquisition was performed using a Leica DM 6000B microscope (Wetzlar, Germany) and MetaMorph software.

Article Title: Porcine CMV Infection Is Associated with Early Rejection of Kidney Grafts in a Pig to Baboon Xenotransplantation Model
Article Snippet: .. Immunohistochemistry was also performed on frozen sections using polyclonal antibodies reactive to ICAM-1, porcine MHC Class II, and IgG (polyclonal rabbit anti-human IgG (Dako, Denmark). ..

Labeling:

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Article Snippet: .. Positive antibody staining was detected using an HRP- or AP-conjugated labeled polymer system (ImmPRESS Staining Kits, Vector Laboratories) and subsequently visualized using Sigma DAB (NeuN and Iba1), Vector DAB (Fibrinogen), Dako DAB+ (ICAM-1 and 3-NT) or Vector Blue (GFAP). .. In vivo free radical detection by dihydroethidium staining Dihydroethidium (Thermo Fisher Scientific, Inc.; Waltham, MA) was administered by intraperitoneal (IP) injection in at 6 μg/g body weight to mice 3 hrs following CCI-TBI.

Incubation:

Article Title: The Impact of Ischemia/Reperfusion Injury on Liver Allografts from Deceased after Cardiac Death versus Deceased after Brain Death Donors
Article Snippet: .. Sections were incubated with monoclonal antibodies neutrophil elastase (NP57, neutrophils), ICAM-1 (6.5B5), Von Willebrand Factor (F8/86) and VCAM-1 (1.4C3) CD3 (UCHT-1, T lymphocytes) (DAKO Ltd, High Wycombe, UK), HLA class II (NFK1, monomorphic HLA-DR, DP)[ ] and P-selectin (AK4), CD41 (MWReg30, anti-platelets) and FasL (G247-4) (Pharmingen, Ltd, San Diego, USA). .. TUNEL staining TUNEL technique was performed to detect apoptosis in situ using R & D systems kit.

Article Title: The Junctional Adhesion Molecule 3 (JAM-3) on Human Platelets is a Counterreceptor for the Leukocyte Integrin Mac-1
Article Snippet: .. After incubation at 22°C for 2 h and a washing step, bound JAM-3 was detected by biotinylated mAb Gi11 and peroxidase-conjugated streptavidin (1:2,000 dilution; Dako), bound ICAM-1 was detected by appropriate mAb and peroxidase-conjugated goat anti–mouse immunoglobulin (1:2,000 dilution; Dako). ..

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Article Snippet: .. Prior to primary antibody incubation, sections stained for GFAP, Iba1 and ICAM-1 were HIER pre-treated with 10 mM citric acid buffer (pH 6.0), while those stained for Fibrinogen were PIER pre-treated with Proteinase K (Dako). .. Sections stained for NeuN received no pre-treatment.

Staining:

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Article Snippet: .. Positive antibody staining was detected using an HRP- or AP-conjugated labeled polymer system (ImmPRESS Staining Kits, Vector Laboratories) and subsequently visualized using Sigma DAB (NeuN and Iba1), Vector DAB (Fibrinogen), Dako DAB+ (ICAM-1 and 3-NT) or Vector Blue (GFAP). .. In vivo free radical detection by dihydroethidium staining Dihydroethidium (Thermo Fisher Scientific, Inc.; Waltham, MA) was administered by intraperitoneal (IP) injection in at 6 μg/g body weight to mice 3 hrs following CCI-TBI.

Article Title: Platelet expression of tumour necrosis factor-alpha (TNF-?), TNF receptors and intercellular adhesion molecule-1 (ICAM-1) in patients with proliferative diabetic retinopathy
Article Snippet: .. To confirm that the staining for TNF-α, TNF-R and ICAM-1 was associated to platelets, we performed dual staining for CD41 and TNF-α or CD41 and TNF-R in several samples using CD41 antibody conjugated with RPE (Dako). ..

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Article Snippet: .. Prior to primary antibody incubation, sections stained for GFAP, Iba1 and ICAM-1 were HIER pre-treated with 10 mM citric acid buffer (pH 6.0), while those stained for Fibrinogen were PIER pre-treated with Proteinase K (Dako). .. Sections stained for NeuN received no pre-treatment.

Affinity Purification:

Article Title: The role for decorin in delayed-type hypersensitivity
Article Snippet: .. For ICAM-1, a secondary peroxidase conjugated affinity purified anti-Rat IgG (Dako, Glostrup, DK) diluted 1/1000 with PBS containing 1% BSA, anti-rabbit IgG (Amersham, Braunschweig, Germany) was applied for 30 minutes at room temperature. .. After washing with PBS, sections were incubated for 5 minutes with DAB reagent (Dako) and hematoxylin counterstained.

Plasmid Preparation:

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury
Article Snippet: .. Positive antibody staining was detected using an HRP- or AP-conjugated labeled polymer system (ImmPRESS Staining Kits, Vector Laboratories) and subsequently visualized using Sigma DAB (NeuN and Iba1), Vector DAB (Fibrinogen), Dako DAB+ (ICAM-1 and 3-NT) or Vector Blue (GFAP). .. In vivo free radical detection by dihydroethidium staining Dihydroethidium (Thermo Fisher Scientific, Inc.; Waltham, MA) was administered by intraperitoneal (IP) injection in at 6 μg/g body weight to mice 3 hrs following CCI-TBI.

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    Agilent technologies icam 1 promoter construct pgl4 icam 1 1 3
    Identification of the DNA binding site involved in Erg-mediated repression of <t>ICAM-1.</t> A , schematic diagram of ICAM-1 promoter mutant constructs. <t>pGL4</t> ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p
    Icam 1 Promoter Construct Pgl4 Icam 1 1 3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/icam 1 promoter construct pgl4 icam 1 1 3/product/Agilent technologies
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    93
    Agilent technologies intercellular adhesion molecule 1
    Identification of the DNA binding site involved in Erg-mediated repression of <t>ICAM-1.</t> A , schematic diagram of ICAM-1 promoter mutant constructs. <t>pGL4</t> ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p
    Intercellular Adhesion Molecule 1, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/intercellular adhesion molecule 1/product/Agilent technologies
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    86
    Agilent technologies icam 1 3 utr plasmid construct
    Identification of the DNA binding site involved in Erg-mediated repression of <t>ICAM-1.</t> A , schematic diagram of ICAM-1 promoter mutant constructs. <t>pGL4</t> ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p
    Icam 1 3 Utr Plasmid Construct, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Identification of the DNA binding site involved in Erg-mediated repression of ICAM-1. A , schematic diagram of ICAM-1 promoter mutant constructs. pGL4 ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The Transcription Factor Erg Controls Endothelial Cell Quiescence by Repressing Activity of Nuclear Factor (NF)-?B p65 *

    doi: 10.1074/jbc.M112.346791

    Figure Lengend Snippet: Identification of the DNA binding site involved in Erg-mediated repression of ICAM-1. A , schematic diagram of ICAM-1 promoter mutant constructs. pGL4 ICAM-1 1.3 was mutated in either single or double EBS or in the NF-κB binding site. EBS were mutated from GGAA to CCAA. NF-κB was mutated as previously shown ( 15 ). B , ICAM-1 promoter activity of EBS mutants after Erg Genebloc treatment. Results are expressed as luciferase activity relative to control Genebloc-treated cells. C , ICAM-1 promoter activity of EBS mutants after AdErg treatment, expressed as luciferase activity relative to AdLacZ ( n = 3–7). *, p

    Article Snippet: Site-directed Mutagenesis of ICAM-1 Promoter Constructs The ICAM-1 promoter construct pGL4 ICAM-1 1.3 containing the first 1.3 kb upstream from the transcription start site as described in Ref. was mutated within ETS binding sites (EBS), or within the NF-κB binding site as previously shown , using the QuikChange® lightning multi site-directed mutagenesis kit (Agilent), all primers were designed using the QuikChange® Primer Design Program (Agilent).

    Techniques: Binding Assay, Mutagenesis, Construct, Activity Assay, Luciferase