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. 2020 Aug 2;57(11):4467–4487. doi: 10.1007/s12035-020-02042-w

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — 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