lamp  (StressMarq)

Journal: GeroScience

Article Title: Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice

doi: 10.1007/s11357-022-00562-y

Figure Lengend Snippet: Microglial phagocytosis of neurons, expression of autophagy markers, and lipofuscin content is chronically increased with age and injury. Phagocytosis was assessed by intracellular detection of neuronal and myelin antigens. A The percentage of NeuN-positive microglia was significantly increased with age and injury. The mean fluorescence intensity of FluoroMyelin red staining was acutely increased in microglia from both age groups (B) . The presence of vesicular glutamate transporter 1 (vGlut1) (C) and the phagosome marker CD68 (D ) were increased in microglia with both old age and TBI. Representative histograms illustrate the relative abundance of lysosomes and LC3II-positive autophagosomes in microglia as measured by LysoTracker (E) and Cyto-ID Autophagosome dyes (F) . For all histograms, gray = FMO control, blue = young, red = old, sham = no outline/no fill, 48 h TBI = bold outline/no fill, and 12w TBI = bold outline/bold fill. The mean fluorescence intensity of Lamp1 (G) and Sqstm1/p62 (H) for microglia are shown. N = 5–7/group. Data were analyzed using 2-way ANOVA group analysis with Tukey’s test for multiple comparisons. **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Intracellular staining for Ki67-PECy7 (Biolegend, Cat# 652426), PCNA-AF647 (Biolegend, Cat# 307912), CD68-PerCPCy5.5 (Biolegend, Cat# 137010), NeuN-PE (Millipore Sigma, Cat# FCMAB317PE), Vglut1-APC (StressMarq, Cat# SMC-394D-APC), Lamp1-PerCPCy5.5 (Biolegend, Cat# 121626), Lamp2-PE (Biolegend, Cat# 108506), Sqstm1/p62-AF647 (Novus Biologicals, Cat# NBP1-42822AF647), ATG5-AF647 (Biolegend, Cat# 847410), ATG7-AF700 (R&D Systems, Cat# FAB6608N), Ubiquitin-AF647 (Biolegend, Cat# 838710), H3-AF647 (Cell Signaling Technology, Cat# 12230S), Acetylated (Ac) Lysine (Lys)-PECy7 (Biolegend, Cat# 623408), H3-Ac-Lys9-AF488 (Cell Signaling Technology, Cat# 9683S), H3-Ac-Lys18-AF488 (Cell Signaling Technology, Cat# 73508S), H3-Ac-Lys27-AF647 (Cell Signaling Technology, Cat# 39030S), H3-Ac-Lys36-AF647 (Cell Signaling Technology, Cat# 84061S), Phospho(ser149)-H2A.X-PECy7 (Biolegend, Cat# 613420), p16-APC (StressMarq, Cat# SPC-1280D-APC), and p21-AF488 (RND-NBP2-43697AF488) was performed using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat# 554714) according to manufacturer’s instructions and as described previously [ ].

Techniques: Expressing, Fluorescence, Staining, Marker

Journal: GeroScience

Article Title: Functional and transcriptional profiling of microglial activation during the chronic phase of TBI identifies an age-related driver of poor outcome in old mice

doi: 10.1007/s11357-022-00562-y

Figure Lengend Snippet: Trehalose treatment reduces long-term microgliosis, lymphocyte infiltration, and phagocytosis of neurons following TBI. A A representative dot plot of leukocyte populations in the brain at 9 weeks after TBI. Quantification of CD45 int CD11b + microglia (B ), CD45 hi CD11b + myeloid cells (C) , and CD45 hi CD11b − putative lymphocyte (D) cell counts are shown. Representative histograms show the relative level of E CD68 protein expression and F LipiBlue-stained lipid bodies in microglia. G The percentage of Vglut1-positive microglia in each treatment group is quantified. H Representative dot plots illustrate the percentage of NeuN-positive microglia in the ipsilateral hemisphere at 8 weeks post-TBI. The mean fluorescence intensity of NeuN immunoreactivity is quantified. For all histograms, gray = FMO control, sucrose (vehicle) treated = blue, trehalose treated = red, sham controls = no fill, and TBI groups = bold fill. N = 9–10/group (A–F) and N = 4–6/group (G–H) . Data were analyzed using 2-way ANOVA group analysis with Tukey’s test for multiple comparisons. **** p < 0.0001, ** p < 0.01, * p < 0.05

Article Snippet: Intracellular staining for Ki67-PECy7 (Biolegend, Cat# 652426), PCNA-AF647 (Biolegend, Cat# 307912), CD68-PerCPCy5.5 (Biolegend, Cat# 137010), NeuN-PE (Millipore Sigma, Cat# FCMAB317PE), Vglut1-APC (StressMarq, Cat# SMC-394D-APC), Lamp1-PerCPCy5.5 (Biolegend, Cat# 121626), Lamp2-PE (Biolegend, Cat# 108506), Sqstm1/p62-AF647 (Novus Biologicals, Cat# NBP1-42822AF647), ATG5-AF647 (Biolegend, Cat# 847410), ATG7-AF700 (R&D Systems, Cat# FAB6608N), Ubiquitin-AF647 (Biolegend, Cat# 838710), H3-AF647 (Cell Signaling Technology, Cat# 12230S), Acetylated (Ac) Lysine (Lys)-PECy7 (Biolegend, Cat# 623408), H3-Ac-Lys9-AF488 (Cell Signaling Technology, Cat# 9683S), H3-Ac-Lys18-AF488 (Cell Signaling Technology, Cat# 73508S), H3-Ac-Lys27-AF647 (Cell Signaling Technology, Cat# 39030S), H3-Ac-Lys36-AF647 (Cell Signaling Technology, Cat# 84061S), Phospho(ser149)-H2A.X-PECy7 (Biolegend, Cat# 613420), p16-APC (StressMarq, Cat# SPC-1280D-APC), and p21-AF488 (RND-NBP2-43697AF488) was performed using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Cat# 554714) according to manufacturer’s instructions and as described previously [ ].

Techniques: Expressing, Staining, Fluorescence