Fig 1: Relative GFAP Expression and Immunoreactive Density Following FUS+MB Exposure. (A) Two days following sonication, the mean ratio of GFAP expression in the sonicated to non-sonicated dorsal hippocampi for saline-treated animals was 1.36 ± 0.26 (p = 0.005). Animals receiving DEX displayed no significant differences (p = 0.56). (B) Ten days post-FUS+MBs, the mean ratio of GFAP immunoreactive density in the sonicated to non-sonicated dorsal hippocampi for saline-treated animals was 1.10 ± 0.07 (p = 0.01). Animals receiving DEX did not display a significant difference in dorsal hippocampal GFAP immunoreactive density between hemispheres (mean ratio of 1.06 ± 0.09 for GFAP immunoreactive density in the sonicated to the non-sonicated dorsal hippocampi; p = 0.11). (C) Representative images of GFAP immunoreactivity 10 days following FUS+MB exposure demonstrating reactive astrocytes in the sonicated hippocampus of saline-treated animals. * indicates p < 0.05, paired student's t-test. Red, horizontal, dashed line indicates no difference between sonicated and non-sonicated dorsal hippocampi. Error bars represent standard error of the mean. n = 7 saline-treated and 7 DEX-treated animals for analysis 2 days post-FUS+MB exposure. n = 7 saline-treated and 8 DEX-treated animals for analysis 10 days post-FUS+MB exposure. Scale bar = 100 µm.
Fig 2: Pretreatment with RvD2 reduced spinal IL-17 secretion, CXCL1 release and astrocyte activation in animals with bone cancer pain. Intrathecal RvD2 (500 ng) was given daily on days 4, 5 and 6 following sarcoma cell implantation. All data from biochemical experiments were collected on day 7 following sham and sarcoma implantations. (A–C) ELISA assay showed that RvD2 pre-treatment down-modulated the elevated levels of spinal IL-17, CXCL1 and GFAP (the marker of astrocyte activation) proteins in sarcoma animals. (D) Immunohistochemistry staining showed representative photomicrographs of spinal GFAP following sarcoma interventions and RvD2 exposure (scale bar, 50 µm). Results from biochemical experiments are presented as the mean ± SEM (n = 4) and compared using one-way ANOVA followed by Bonferroni multiple comparisons. # p < 0.05 vs. group sham + vehicle. * p < 0.05 vs. group sarcoma + vehicle.
Fig 3: In vivo and in vitro evidence demonstrates that astrocytes (in the brain, spinal cord and retina) and Müller glia (in the retina) are capable of engulfing microglial debris.a Scheme of the in vivo astrocytic engulfment examination by ALDH1L1-CreER::Ai14-based astrocyte labeling and microglial depletion. Because ALDL1L1-CreER::Ai14 does not target retinal astrocytes, retinal astrocytes are visualized by GFAP in C57BL/6 J mice treated with CD or PLX5622 for 2 days. b, c Confocal orthogonal colocalization (b) and 3D reconstruction (c) show that tdTomato+ astrocytes (in the brain, spinal cord and retina) or Müller glia (in the retina) do not engulf IBA1+ microglial debris under physiological conditions (D17) whereas they engulf IBA1+ microglial debris upon the CSF1R inhibition (D19). d Quantification of microglial debris engulfment by astrocytes (in the brain, spinal cord and retina) and Müller glia (in the retina). N = 6 mice at D17, 5 mice at D19. Two-tailed independent t test. e Scheme of the in vitro astrocytic engulfment assay using pHrodo-labeled microglial debris. f GFAP+ astrocytes engulf pHrodo-labeled microglial debris in FBS-containing culture medium. pHrodo is illustrated by the green pseudocolor for better visualization. g Quantification of astrocytic engulfment after exposure to pHrodo-labeled microglial debris for 0, 24, 48, and 72 h. N = 5 independent biological replicates for each group. One-way ANOVA with Holm?Sidak’s multiple comparisons test (post hoc). PLX5622: PLX5622-formulated AIN-76A diet; CD: control AIN-76A diet; PLX: PLX5622; MFI mean fluorescence intensity. Data are presented as mean ± SD. The source data are provided as a Source Data file.
Fig 4: Microglial debris is unlikely to be engulfed by pericytes, endothelial cells, VSMCs, OPCs, oligodendrocytes, NSCs, neurons or circulating blood cells in vivo.a Scheme of in vivo debris engulfment examinations by microglial depletion. b Microglial debris is unlikely to be phagocytosed by pericytes, endothelial cells, VSMCs, OPCs, oligodendrocytes, neurons or NSCs in vivo. NESTIN-GFP is illustrated by the red pseudocolor for better visualization. Astrocytes (GFAP): 0.24 ± 0.05%, pericytes (PDGFR-ß): 0.03 ± 0.01%, endothelial cells (CD31): 0.03 ± 0.01%, VSMCs (a-SMA): 0.03 ± 0.02%, OPCs (PDGFR-a): 0.08 ± 0.02%, oligodendrocytes (MBP): 0.05 ± 0.01%, oligodendrocytes (CC1): 0.07 ± 0.02%, neurons (TUJ1): 0.04 ± 0.01%, NSCs (NESTIN-GFP): 0.03 ± 0.01%. N = 4 mice in the NESTIN-GFP group and N = 5 mice in the rest of the groups. One-way ANOVA with Holm?Sidak’s multiple comparisons test (post hoc). P = 3.1086e–015, 4.6629e–015, 2.6645e–015, 1.0612e–012, 3.4639e–014, 9.7167e–013, 4.9960e–015, and 1.2434e–014. c Scheme of the in vivo BBB integrity examination with 10-kDa dextran-FITC. d Confocal images show that the BBB is not compromised during microglial depletion in vivo. Each experiment was independently repeated from 4 mice with similar results. PLX5622: PLX5622-formulated AIN-76A diet; VSMCs vascular smooth muscle cells, OPC oligodendrocyte precursor cell, OL oligodendrocyte, NSC neural stem cell, IV intravenous. The source data are provided as a Source Data file.
Fig 5: Pretreatment with RvD2 reduced spinal IL-17 secretion, CXCL1 release and astrocyte activation following peripheral nerve trauma. Intrathecal RvD2 (500 ng) was administered daily on days 4, 5 and 6 following CCI operations. All biochemical data were collected on day 7 following sham and CCI interventions. (A–C) ELISA assays showed that RvD2 pretreatment down-modulated the elevated levels of spinal IL-17, CXCL1 and GFAP (the marker of astrocyte activation) proteins in CCI animals. (D) Immunohistochemistry staining showed representative photomicrographs of GFAP in the spinal dorsal horn following CCI surgeries and RvD2 exposure (scale bar, 50 µm). Results from biochemical experiments are presented as the mean ± SEM (n = 4) and compared using one-way ANOVA followed by Bonferroni multiple comparisons. # p < 0.05 vs. group sham + vehicle. * p < 0.05 vs. group CCI + vehicle.
Supplier Page from Abcam for Mouse GFAP ELISA Kit