application measure colocalization  (MetaMorph Inc)

Journal: Molecular Neurobiology

Article Title: A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia

doi: 10.1007/s12035-020-02042-w

Figure Lengend Snippet: Immunocytochemistry and confocal imaging of TSPO colocalization with gp91 phox , p22 phox , VDAC, and LAMP-2 and effect of LPS activation in primary microglia. a Triple-label immunofluorescent confocal images of TSPO colocalization with gp91 phox , p22 phox , or VDAC in primary microglia (Mac-1 labeled) cells. Confocal images show that TSPO colocalizes with gp91 phox , p22 phox , and VDAC in primary microglia. Images in upper panels are at a low magnification: scale bar = 40 μm. Cells in white boxes were selected to be expressed at a higher magnification: scale bar = 10 μm (lower panel). b Analyses of protein pair signal colocalization revealed that TSPO has a high degree of colocalization with gp91 phox (64.68%), p22 phox (72.82%), and VDAC (81.48%) and a lower level of colocalization (20.13%) with the lysosomal marker, LAMP-2 in vehicle-treated microglia. Analysis of variance with Tukey’s post hoc tests shows a significant effect of protein colocalization with TSPO ( F 3,6 = 9.8; p = 0.0006) where TSPO colocalization with LAMP-2 is significantly lower than with gp91 phox , p22 phox , and VDAC (* = p < 0.01). c The percentage of gp91 phox , p22 phox , and VDAC that colocalized with TSPO decreased when microglia were activated with 100 ng/mL of LPS for 18 h relative to vehicle conditions (% gp91 with TSPO: p = 0.004; %p22 with TSPO: p = 0.002; % VDAC with TSPO: p = 0.003). These results indicate that microglia activation disrupts TSPO’s association with gp91 phox , p22 phox , and VDAC. d Further analysis of signal colocalization indicates that under LPS-stimulated conditions, TSPO associated with gp91 and TSPO associated with p22, exhibit significantly decreased colocalization with VDAC suggesting a movement from the mitochondria to other cellular compartments (% (TSPO with gp91)/ VDAC: p < 0.001. % (TSPO with p22) / VDAC: p = 0.004). Data are expressed as mean ± s.e.m. n = 5–7 independent experiments with > 35 microglia counted per treatment condition per experiment. n = 3 independent experiments for TSPO/LAMP-2 labeling with > 35 microglia counted per treatment condition per experiment. Student’s paired t test was performed; * p < 0.05 compared to vehicle-treated microglia

Article Snippet: For colocalization analyses, gray scale images of each wavelength (each protein) were used to examine the area of colocalized pixels of both wavelengths to calculate the percent colocalization of their signals using the Metamorph Application “Measure Colocalization.” Percent colocalization was calculated as previously described [ ].

Techniques: Immunocytochemistry, Imaging, Activation Assay, Labeling, Marker

Journal: Molecular Neurobiology

Article Title: A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia

doi: 10.1007/s12035-020-02042-w

Figure Lengend Snippet: Proximity ligation assay (PLA) supports a TSPO interaction with gp91 phox , p22 phox , and VDAC in primary microglia. a – c Representative PLA phase and pseudo-colored confocal images of protein pair interactions (colored dots) in microglia: TSPO + gp91 ( a ); TSPO + p22 ( b ); TSPO + VDAC ( c ). The images indicate that TSPO interacts with gp91 phox , p22 phox , and VDAC respectively supporting the co-immunoprecipitation and immunocytochemistry colocalization results. d Quantification of average number of protein pair signals per microglia in vehicle conditions at 18 h. PLA experiments confirms that TSPO interacts with the NOX2 subunits gp91 phox and p22 phox , as well as the mitochondrial protein, VDAC. Imaging and quantification included negative controls for labeling conditions of single antibody only, as well as no primary antibody, to ensure signal specificity. Data are expressed as mean ± s.e.m. n = 4–7 independent experiments with > 30 cells counted per treatment and per labeling condition. One-way ANOVA across labeling conditions was performed; (TSPO+gp91: F 3,17 = 8.193, p = 0.002; TSPO+p22: F 3,11 = 11.108, p = 0.003; TSPO+VDAC: F 3,14 = 9.979, p = 0.002). * p < 0.05 = significantly different relative to all other conditions within a protein pair group

Article Snippet: For colocalization analyses, gray scale images of each wavelength (each protein) were used to examine the area of colocalized pixels of both wavelengths to calculate the percent colocalization of their signals using the Metamorph Application “Measure Colocalization.” Percent colocalization was calculated as previously described [ ].

Techniques: Proximity Ligation Assay, Immunoprecipitation, Immunocytochemistry, Imaging, Labeling

Journal: Molecular Neurobiology

Article Title: A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia

doi: 10.1007/s12035-020-02042-w

Figure Lengend Snippet: In situ colocalization of TSPO with gp91 phox in murine brain microglia. a Representative triple labeled immunofluorescent confocal images in the thalamus of a 2-month old Sandhoff disease mouse at 60× with 1.6× zoom. Imaging confirmed that the TSPO signal colocalizes with the gp91 phox signal in Mac-1 labeled microglia. b Quantification of percent colocalization of TSPO with gp91 phox in microglia in the thalamus of Sandhoff disease mice compared to wildtype as a function of age. The data indicates that the TSPO-gp91 phox interaction increases as a function of age and progression of neurological disease (age: F 3,7 = 4.257, p = 0.017; genotype: F 1,7 = 11.081, p = 0.003; age × genotype: F 3,7 = 1.548, p = 0.232). Data are expressed as mean ± s.e.m. n = 4 independent animals and experiments per group, except for the 2-month old wildtype group which has n = 3 due to one animal being excluded as an outlier (> 3 standard deviations from the mean)

Article Snippet: For colocalization analyses, gray scale images of each wavelength (each protein) were used to examine the area of colocalized pixels of both wavelengths to calculate the percent colocalization of their signals using the Metamorph Application “Measure Colocalization.” Percent colocalization was calculated as previously described [ ].

Techniques: In Situ, Labeling, Imaging