fa 11  (Bio-Rad)

Journal: bioRxiv

Article Title: FcγR- and CD9-dependent synapse engulfing microglia in the thalamus drives cognitive impairment following cortical brain injury

doi: 10.1101/2024.09.19.609743

Figure Lengend Snippet: (A) Representative images of Iba1 + microglia in the cortex (primary lesion) and the thalamus 3 and 21 days after unilateral cortical ablation injury and sham surgery. MD, mediodorsal nucleus; CM, central medial nucleus; VPL, ventral posterolateral nucleus; VPM, Ventral posteromedial nucleus. Scale bar, 500 µm. (B) Quantification data of the area covered by Iba1 + microglia (%) in the cortex and thalamus (n = 3-4 mice for sham; n = 3-7 mice for injury). (C) Representative images of TNF-α and CD68 expression in Iba1 + microglia in the thalamus 21 days after cortical brain injuries and sham surgery. Scale bar, 10 µm. (D) Quantification data of the number of TNF-α expressing Iba1 + microglia and average CD68 + area in Iba1 + microglia (n = 3 mice for sham; n = 3 mice for injury). (E) Representative images of PSD95 + signals overlapped with CD68 + area in Iba1 + microglia of the thalamus after cortical brain injuries and sham surgery. Scale bar: 10 µm. PSD95 + signals colocalized to CD68 + lysosomal area per Iba1 + microglia (PSD95 + CD68 + area inside Iba1 + microglia) were calculated per image, averaged across multiple brain sections per mouse and shown in bar graphs for the sham (n = 4 mice) and the injury (n = 4 mice) group. (F) Representative images of PSD95 post-synaptic proteins in the thalamus after cortical brain injuries and sham surgery. Scale bar: 10 µm. PSD95 + puncta density was calculated in the thalamus after cortical brain injuries and sham surgery. (n = 4 mice for sham; n = 4 mice for injury). (G) Experimental timeline for novel object recognition (NOR) test after cortical brain injuries. (H) Discrimination index in the NOR test for injury (n = 6) and sham (n = 7 mice) group. The discrimination index was calculated as ([time spent at the novel object] – [time spent at the familiar object])/([time spent at the novel object] + [time spent at the familiar object]). (I) Correlation plot of the discrimination index and thalamic Iba1 intensity 21 days after injuries and sham surgery (n = 7 mice). (J) Representative images of neuronal c-Fos expression in the thalamus, PRh, mPFC, and hippocampus (CA1, CA3) 21 days after injuries and sham surgery. Scale bars, 100 µm. (K) Quantification data of neuronal c-Fos expression in the injury group (n = 6-7 mice) relative to the sham group (n = 3-4 mice). (L) Experimental timeline for neuronal DREADD rescue experiment. AAV-hSyn-hM3D (Gq)-mCherry was injected into the thalamus on the day of cortical brain injuries, and CNO or vehicle was administered intraperitoneally before the test session in the NOR test. (M) Quantification data of neuronal c-Fos expression in the CNO-treated group (n = 8 mice) relative to the vehicle-treated mice (n = 11 mice). (N) Discrimination index in the NOR test for vehicle-treated (n = 11 mice) and CNO-treated mice (n = 8 mice). In bar graphs, data are shown as the mean ± s.e.m, and each dot represents an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001; Student’s t -test (B, D, F, K, M, and N) and one-way ANOVA with post hoc Dunnett’s test (I). See also Figures S1-S3.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-Iba1 (1:500; WAKO, #019–19741,), Alexa Fluor 488-conjugated rabbit monoclonal anti-Iba1 (1:500; abcam, #ab225260), mouse monoclonal anti-Iba1 (1:500; Sigma-Aldrich, #MABN92), Alexa Fluor 647-conjugated mouse monoclonal anti-NeuN (1:500; abcam, #ab190565), rabbit monoclonal anti-NeuN (1:500; abcam, #ab177487), rabbit monoclonal anti-c-Fos (1:500; Cell Signaling Technology, #2250), rabbit monoclonal anti-TSPO (1:100; abcam, #ab109497), rat monoclonal anti-CD68 (1:2,000; Novus Biologicals, #NBP2-33337), mouse monoclonal anti-TNF-α (1:100; abcam, #ab1793), rat monoclonal anti-RFP (1:500; Chromotek, #5F8), rabbit monoclonal anti-HA-tag (1:500; Cell Signaling Technology, #3724), armenian hamster monoclonal anti-CD31/PECAM1 (1:100; DSHB, #2H8-s), rabbit polyclonal CD9 (1:100; Proteintech, #20597-1-AP), CD9 (1:100; BD Biosciences, #553758), rabbit polyclonal anti-FcγRIII (1:100; abcam, #ab203883), rabbit polyclonal anti-p-Syk (1:200; Cell Signaling, #2710T), mouse monoclonal anti-PSD95 (1:500; Thermo Fisher Scientific, #MA1-046), goat polyclonal anti-LPL (1:100; R&D Systems, #AF7197), chicken monoclonal anti-Tmem119 (1:500; Synaptic Systems, #400009), sheep polyclonal anti-Trem2 (1:100; R & D, #AF1729), rabbit polyclonal anti-Claudin5 (1:100, Thermo Fisher Scientific, #34-1600), and rabbit polyclonal anti-Ifitm3 (1:100; Proteintech, #11714-1-AP).

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: FcγR- and CD9-dependent synapse engulfing microglia in the thalamus drives cognitive impairment following cortical brain injury

doi: 10.1101/2024.09.19.609743

Figure Lengend Snippet: (A) Experimental timeline for blocking CD9 by anti-CD9 Ab injection. (B) Representative images of PSD95 + signals overlapped with CD68 + lysosomal area of Iba1 + microglia in the thalamus of control Ab-and anti-CD9 Ab-injected mice after cortical brain injuries. PSD95 + puncta colocalized to CD68 + lysosomal area in Iba1 + microglia (PSD95 + CD68 + Iba1 + area) are calculated per animal and shown in bar graphs for the control Ab-(n= 9 mice) and the anti-CD9 Ab-injected group (n = 10 mice). Scale bar: 10 µm. (C) Discrimination index in the NOR test for the control Ab (n = 9 mice) and the anti-CD9 Ab group (n = 10 mice). (D) UMAP plot highlighting 6 microglia clusters after cortical brain injuries in control Ab-injected group thalamus, in anti-CD9 Ab-injected group thalamus, and in thalamus from , and bar graphs showing the percentages of each microglial cluster in control Ab group (red) and in anti-CD9 Ab group (blue), with the percentages reaching 100% for each group. (E) Pseudo-bulk heat map showing log2FC of mean gene expression in thalamic MG#1 between the anti-CD9 Ab group and control Ab group, highlighting up-or down-regulated genes that are Syk-dependent or enriched in MG#2/3, MG#4, and MG#6. Positive values indicate higher expression (red) and negative values indicate lower expression in (blue). (F) A schematic diagram showing how MG#2 and #3 transit to MG#4, #5, and #6 via MG#1, and how anti-CD9 Ab acts on this pathway. (G) Representative images of p-Syk signals in Iba1 + microglia of the thalamus in control Ab-and anti-CD9 Ab-injected group after cortical brain injuries. Quantification data of the p-Syk levels in Iba1 + microglia in the anti-CD9 Ab group (n = 10 mice) relative to control Ab group (n = 9 mice) are shown in bar graphs. Scale bar: 10 µm. (H) Volcano plot of differentially expressed genes in the thalamic MG#4 microglia in anti-CD9 Ab group compared to the thalamic MG#4 microglia in control Ab group. Red: genes downregulated in anti-CD9 Ab group. Blue: genes upregulated in anti-CD9 Ab group. In bar graphs, data are shown as the mean ± s.e.m, and each dot represents an individual animal. ** p < 0.01, *** p < 0.001; Student’s t -test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-Iba1 (1:500; WAKO, #019–19741,), Alexa Fluor 488-conjugated rabbit monoclonal anti-Iba1 (1:500; abcam, #ab225260), mouse monoclonal anti-Iba1 (1:500; Sigma-Aldrich, #MABN92), Alexa Fluor 647-conjugated mouse monoclonal anti-NeuN (1:500; abcam, #ab190565), rabbit monoclonal anti-NeuN (1:500; abcam, #ab177487), rabbit monoclonal anti-c-Fos (1:500; Cell Signaling Technology, #2250), rabbit monoclonal anti-TSPO (1:100; abcam, #ab109497), rat monoclonal anti-CD68 (1:2,000; Novus Biologicals, #NBP2-33337), mouse monoclonal anti-TNF-α (1:100; abcam, #ab1793), rat monoclonal anti-RFP (1:500; Chromotek, #5F8), rabbit monoclonal anti-HA-tag (1:500; Cell Signaling Technology, #3724), armenian hamster monoclonal anti-CD31/PECAM1 (1:100; DSHB, #2H8-s), rabbit polyclonal CD9 (1:100; Proteintech, #20597-1-AP), CD9 (1:100; BD Biosciences, #553758), rabbit polyclonal anti-FcγRIII (1:100; abcam, #ab203883), rabbit polyclonal anti-p-Syk (1:200; Cell Signaling, #2710T), mouse monoclonal anti-PSD95 (1:500; Thermo Fisher Scientific, #MA1-046), goat polyclonal anti-LPL (1:100; R&D Systems, #AF7197), chicken monoclonal anti-Tmem119 (1:500; Synaptic Systems, #400009), sheep polyclonal anti-Trem2 (1:100; R & D, #AF1729), rabbit polyclonal anti-Claudin5 (1:100, Thermo Fisher Scientific, #34-1600), and rabbit polyclonal anti-Ifitm3 (1:100; Proteintech, #11714-1-AP).

Techniques: Blocking Assay, Injection, Control, Expressing

Journal: bioRxiv

Article Title: FcγR- and CD9-dependent synapse engulfing microglia in the thalamus drives cognitive impairment following cortical brain injury

doi: 10.1101/2024.09.19.609743

Figure Lengend Snippet: (A) Representative images of IgG and CD31 + blood vessels of the thalamus and the HPC after cortical brain injuries. Percentages of extravascular IgG coverage area per total IgG coverage area are shown in bar graphs for the HPC (n = 4 mice) and the thalamus (n = 4 mice) group. (B) Representative images of Claudin5 and CD31 + blood vessels in the hippocampus (HPC) and the thalamus after cortical brain injuries. Quantification of the relative expression of Claudin5 in CD31 + blood vessels in the HPC (n = 4 mice) and the thalamus (n = 4 mice) is shown in bar graphs. (C) Representative images of IgG and CD31 + blood vessels in the thalamus of cortically injured mice treated with control Ab and anti-CD9 Ab. Percentages of extravascular IgG coverage area per total IgG coverage area are shown in bar graphs for the control Ab (n = 10 mice) and the anti-CD9 Ab (n = 9 mice) group. (D) Experimental timeline for blocking Fcγ receptor III (FcγRIII) by anti-FcγRIII Ab injection. (E) Representative images of CD9 hi Iba1 + thalamic microglia in the cortically injured mice injected with control Ab and anti-FcγRIII Ab. Quantification data are shown in bar graphs for the control Ab (n = 10 mice) and the anti-FcγRIII Ab group (n = 9 mice). (F) Representative images of PSD95 + signals overlapped with CD68 + lysosomal area in Iba1 + microglia of the thalamus in control Ab and anti-FcγRIII Ab injected mice after cortical brain injuries. PSD95 + signals in CD68 + lysosomes per Iba1 + microglia are calculated as PSD95 + CD68 + area and shown in bar graphs for control Ab (n= 10 mice) and anti-FcγRIII Ab group (n = 9 mice). (G) Representative images of PSD95 + synaptic puncta of the thalamus in control Ab-and anti-FcγRIII Ab-injected mice after cortical brain injuries. Quantification data are shown in bar graphs for control (n = 10 mice) and anti-CD9 Ab-injected group (n = 9 mice). (H) Discrimination index in the NOR for control Ab (n = 10 mice) and anti-FcγRIII Ab (n = 9 mice) group. (I) Graphic illustration showing how synapse-engulfing microglia develop in response to extravasated IgG via FcγRIII-CD9 signaling, engulfing synapses and impairing neuronal function. This illustration was created with help of BioRender.com. Scale bars, 10 µm (A, E, F, and G) and 100 µm (C). In bar graphs, data are shown as the mean ± s.e.m, and each dot represents an individual animal. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s., not significant; Student’s t -test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-Iba1 (1:500; WAKO, #019–19741,), Alexa Fluor 488-conjugated rabbit monoclonal anti-Iba1 (1:500; abcam, #ab225260), mouse monoclonal anti-Iba1 (1:500; Sigma-Aldrich, #MABN92), Alexa Fluor 647-conjugated mouse monoclonal anti-NeuN (1:500; abcam, #ab190565), rabbit monoclonal anti-NeuN (1:500; abcam, #ab177487), rabbit monoclonal anti-c-Fos (1:500; Cell Signaling Technology, #2250), rabbit monoclonal anti-TSPO (1:100; abcam, #ab109497), rat monoclonal anti-CD68 (1:2,000; Novus Biologicals, #NBP2-33337), mouse monoclonal anti-TNF-α (1:100; abcam, #ab1793), rat monoclonal anti-RFP (1:500; Chromotek, #5F8), rabbit monoclonal anti-HA-tag (1:500; Cell Signaling Technology, #3724), armenian hamster monoclonal anti-CD31/PECAM1 (1:100; DSHB, #2H8-s), rabbit polyclonal CD9 (1:100; Proteintech, #20597-1-AP), CD9 (1:100; BD Biosciences, #553758), rabbit polyclonal anti-FcγRIII (1:100; abcam, #ab203883), rabbit polyclonal anti-p-Syk (1:200; Cell Signaling, #2710T), mouse monoclonal anti-PSD95 (1:500; Thermo Fisher Scientific, #MA1-046), goat polyclonal anti-LPL (1:100; R&D Systems, #AF7197), chicken monoclonal anti-Tmem119 (1:500; Synaptic Systems, #400009), sheep polyclonal anti-Trem2 (1:100; R & D, #AF1729), rabbit polyclonal anti-Claudin5 (1:100, Thermo Fisher Scientific, #34-1600), and rabbit polyclonal anti-Ifitm3 (1:100; Proteintech, #11714-1-AP).

Techniques: Expressing, Control, Blocking Assay, Injection

Journal: Cell reports

Article Title: SIRT2 suppresses aging-associated cGAS activation and protects aged mice from severe COVID-19

doi: 10.1016/j.celrep.2025.115562

Figure Lengend Snippet: Young (5 months old) or old (24 months old) mice were inoculated intranasally with control or ORF3a-expressing AAV6. Mice were analyzed 3 weeks later. n =2 independent experiments. (A and B) Quantitative real-time PCR analyses of the mRNA levels of the indicated genes in BALF cells of young and old mice. n = 8. (C) Quantitative real-time PCR analyses of the mRNA levels of IFNβ in the lungs of young mice. n = 6. (D and E) H&E staining (D) and quantification (E) of lung sections of young mice. Scale bar: 100 μm n = 6 mice/group, 10–15 images examined from 3 slides/mouse. (F) Flow cytometry analysis of myeloid cells (Gr1 + CD11b + ) in BALF of young mice. n = 6. (G and H) Immunostaining for Ly6G + cells (G) and quantification (H) of lung sections. Scale bar: 100 μm n = 8 mice/group, 6 images examined from 3 slides/mouse. (I and J) Immunostaining for CD68 + cells (I) and quantification (J) of lung sections. Scale bar: 100 μm n = 8 mice/group, 4 images examined from 3 slides/mouse. (K and L) Sirius red staining (K) and quantification (L) of lung sections. Scale bar: 200 μm n = 6 mice/group, 10 images examined from 3 slides/mouse. (M and N) Immunostaining for phosphorylated STING (M) and quantification (N) of lung sections of young and old mice. Scale bar: 30 μm n = 8 mice/group, 5 images examined from 3 slides/mouse. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. See also – .

Article Snippet: CD68 clone FA-11 Rat monoclonal antibody , Bio-Rad , Cat# MCA1957GA; RRID:AB_324217.

Techniques: Control, Expressing, Real-time Polymerase Chain Reaction, Staining, Flow Cytometry, Immunostaining