Journal: BMC Cancer

Article Title: Connexin-43 can delay early recurrence and metastasis in patients with hepatitis B-related hepatocellular carcinoma and low serum alpha-fetoprotein after radical hepatectomy

doi: 10.1186/1471-2407-13-306

Figure Lengend Snippet: Relationship among expressions of MVD - CD105 , VEGF , and Cx43 in HCC specimens with serum AFP of < 400 μg / l

Article Snippet: Rabbit anti-human Cx43, VEGF, and CD105 monoclonal antibodies as well as immunohistochemical detection reagents were purchased from ZSGB-Bio Company (Beijing, China).

Techniques:

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Cx43 (gene name: GJA1 ) expression from bulk RNA-sequencing of the human dorsolateral prefrontal cortex in the ROSMAP cohort was plotted against the Braak score, revealing a positive correlation. Right: expression of Cx43 in healthy, mild cognitive impairment (MCI), and AD group. (B) Cx43 protein level from a proteomic study of a multicenter cohort (Baltimore Longitudinal Study of Aging (BLSA), Banner Sun Health Research Institute (Banner), Mount Sinai School of Medicine Brain Bank (MSSB), Adult Changes in Thought Study (ACT)) was plotted against the Braak score. Right: Cx43 protein level in control, asymptomatic AD (asymAD), and AD group. (C) Representative imaging of IBA1 and Aβ immunostaining of healthy controls and AD patients, scale bar: 10 μm. Right: Quantification of IBA1+ microglia number. N = 5 subjects. (D) Representative images of IBA1, GFAP, and Aβ immunostaining of AD patients, scale bar: 10 μm. Right: Distribution of microglia and astrocytes at different distances from Aβ plaques. Cells around total of 30 plaques were calculated. (E) Immunostaining of Cx43 and Aβ, scale bar: 10 μm. Cx43 staining intensity at different distances from Aβ plaques (n=23) was quantified and compared to the area distal to plaque (55-100 μm). (F) Representative images of Cx43, IBA1, and Aβ immunostaining of healthy controls and AD patients, scale bar: 10 μm. The area within white boxes was 3D rendered by Imaris, displaying Cx43 signal in IBA1+ domain. Arrowhead: Periplaque microglia. (G) Quantification of Cx43+ area within IBA1+ domain in normal controls and AD patients, normalized to the mean of normal controls. N = 5 subjects, n = 145 (control) and 207 (AD) cells. (H) Quantification of Cx43+ area within IBA1+ domain in periplaque microglia and distal microglia in AD patients, normalized to the mean of normal controls. N = 5 subjects, n = 85 (periplaque) and 121 (distal) cells. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Expressing, RNA Sequencing Assay, Control, Imaging, Immunostaining, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Volcano plot highlighting differential expressed genes in the ROSMAP-DLPFC-bulk RNA-seq dataset. Cx43/ GJA1 is among the upregulated genes in AD patients. (B) Information on human samples used in this project. (C) Immunostaining of Panx1, IBA1, and Aβ of human samples, and 3D reconstruction of Panx1 signals within IBA1+ domain. Scale bar: 50 μm. Below: quantification of Panx1+ area in microglia normalized to the controls. N = 5 subjects. (D) Immunostaining of Cx43, GFAP, and Aβ of human samples, and 3D reconstruction of Cx43 signals within GFAP+ astrocyte domain. Scale bar: 10 μm. Below: quantification of Cx43+ area in astrocyte normalized to the controls. N = 5 subjects. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: RNA Sequencing Assay, Immunostaining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Representative images of Cx43 and Aβ immunostaining in 9-month-old wild type (WT) and APP/PS1 mice, scale bar: 100 μm. Right: Quantification of Cx43 intensity in the hippocampus of WT and APP/PS1 mice at 2-, 4-, and 9-month-old. N = 5 mice. (B) Representative images of Cx43 and Aβ immunostaining in 4- and 9-month-old APP/PS1 mice, scale bar: 10 μm. Cx43 staining intensity at different distances from Aβ plaques (n = 23) was quantified and compared to the area distal to plaque (50-55 μm). (C) Representative super-resolution image of Cx43, IBA1, and Aβ staining in 4- and 9-month-old WT and APP/PS1 mice, accompanied by 3D reconstruction of Cx43 signal within IBA1+ volume. Scale bar: 10 μm. (D) Quantification of Cx43+ area within IBA1+ domain in WT and APP/PS1 mice, normalized to that of 2-month-old WT. N >80 cells from 5 mice. (E) Comparison of Cx43+ area within IBA1+ domain between periplaque and distal microglia. N > 40 cells from 5 mice. (F) Representative images of Ethidium bromide (EtBr) uptake by 9-month-old APP/PS1 acute brain slices and counterstained with IBA1. Cx43-specific channel blocker GAP26 and pan gap junction/hemichannel blocker CBX were added to 9-month-old APP/PS1 acute brain slices. Scale bar: 10 μm. (G) Quantification of EtBr dye intensity in dye uptake experiment on 2-, 4- and 9-month-old WT and APP/PS1 acute brain slices. N ≥ 5 experiments. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Immunostaining, Staining, Comparison

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) APP/PS1 mice were mated with Cx3cr1-CreERT and Cx43-flox/flox mice to achieve Cx43 conditional knockout in microglia (Cx43 mgl-cKO ). Non-flox or non-Cre APP/PS1 littermates were used as control. (B) Representative images of Methoxy XO4 staining in 9-month-old APP/PS1 and APP/PS1:Cx43 mgl-cKO mouse hippocampus. Scale bar, 50 μm. The percentage of Methoxy X04+ area and the percentage of compact Methoxy X04+ plaque were quantified. N = 5 animals. (C) Representative images of Aβ labelling by 6E10 and dystrophic neurites labelling by RTN3. Scale bar: 10 μm. The number of RTN3+ puncta at different distances from the plaque center and the sum number were quantified. N = 32 plaques. (D) Representative images of Aβ labelling by Methoxy X04 and dystrophic neurites labelling by LAMP1. Scale bar: 10 μm. LAMP1+ area % at different distances from plaque and the total area per plaque were quantified. N = 32 plaques. (E) Representative images of Aβ and postsynaptic element labelling by Homer1. Scale bar: 10 μm. Homer1+ puncta area percentage at different distances from plaque was quantified. N = 32 plaques. (F) MitoSox Red staining assay was performed on acute brain slices to analyze hippocampal CA1 neuronal oxidative stress. Scale bar, 50 μm. MitoSox intensity was quantified and normalized to the WT control of each experiment. N = 5 animals. (G) Novel object recognition test was performed on WT (N = 21), Cx43 mgl-cKO (N = 12), APP/PS1 (N = 14), APP/PS1:Cx43 mgl-cKO (N = 8) mice to examine their cognitive function, and the discrimination index was quantified. (H) Open field test was performed to examine the anxiety traits and locomotor function. Distance spent in the center zone and the total distance were quantified. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Knock-Out, Control, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Super-resolution image of IBA1, Cx43, and Aβ immunostaining of 9-month-old APP/PS1 and APP/PS1:Cx43 mgl-cKO mouse hippocampus, accompanied with 3D reconstruction of Cx43 signal within IBA1+ domain. Scale bar: 10 μm. Cx43+ signal area within microglia was quantified to verify the success of Cx43 knockout. N = 5 animals. (B) Representative image of myelin marker MBP staining. The MBP+ area was quantified. N = 5 animals. (C) Representative image of astrocytic marker GFAP staining. The GFAP+ area and GFAP+ cell number were quantified. N = 5 animals. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Immunostaining, Knock-Out, Marker, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Representative image of IBA1, Aβ and DAPI staining of mouse hippocampus. (B) The total number of IBA1+ microglia, periplaque microglia, and distal microglia was quantified and normalized to the area. N = 5 animals. (C) Representative image of IBA1, Aβ and DAPI staining. For each plaque, the number of periplaque microglia was quantified, and normalized to the plaque perimeter. N = 5 animals. (D) 3D reconstruction image of panel (C) displaying Aβ plaque and the fraction of Aβ plaque within IBA1+ microglia domain. The ratio between plaque within microglia and total plaque volume was quantified. N = 5 animals. (E) Dye uptake experiment was performed on acute brain slices and counterstained with IBA1 to analyze the microglial hemichannel opening. EtBr intensity was quantified and normalized to the WT group for each experiment. N = 5 animals. (F) ELISA analysis of TNFα and IL1β cytokine level in acute brain slices normalized to protein content. (G) Bulk RNA-seq was performed on microglia isolated from WT, APP/PS1, and APP/PS1:Cx43 mgl-cKO mouse cortex and hippocampus. (H) Volcano plot highlighting the differentially expressed gene between APP/PS1:Cx43 mgl-cKO microglia and APP/PS1. (I) Heatmap shows expression of disease associated microglia 3 (DAM3) enriched genes, which were split into 5 clusters according to their expression change in APP/PS1:Cx43 mgl-cKO microglia compared to APP/PS1. Left: normalized gene expression level of 4 clusters. Right: functional annotation and representative genes of 4 clusters. (J-K) Heatmap shows differentially expressed genes that are associated with Aβ recognition, as well as the neuroprotective or neurotoxic signature of microglia. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Isolation, Expressing, Functional Assay

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) The number of periplaque microglia of each plaque was plotted against the plaque perimeter, and the linear regression with 95% confidence interval was shown. N = 76 (APP/PS1), 106 (APP/PS1:Cx43 mgl-cKO ) cells. Related to . (B) The fraction of Aβ plaque within the microglia domain was plotted against plaque volume with the linear regression and 95% confidence interval shown. N = 63 (APP/PS1), 78 (APP/PS1:Cx43 mgl-cKO ) cells. (C) Left: Sholl analysis of periplaque microglia. Right: Sholl analysis of distal microglia and WT microglia. N = 83 (WT), 152 (APP/PS1-periplaque), 224 (APP/PS1:Cx43 mgl-cKO -periplaque), 77 (APP/PS1-distal), 79 (APP/PS1:Cx43 mgl-cKO -distal) cells. (D) Representative images of dye uptake experiment performed on 9-month-old APP/PS1 acute brain slices. Cx43-specific channel blocker GAP26 and pan gap junction/hemichannel blocker CBX were added to 9-month-old APP/PS1 acute brain slices. Scale bar: 10 μm. (E) Heatmap shows differentially expressed genes across WT, APP/PS1, and APP/PS1:Cx43 mgl-cKO microglia. Right: functional annotation analysis of APP/PS1 or APP/PS1:Cx43 mgl-cKO module, i.e., genes significantly enriched in APP/PS1 or APP/PS1:Cx43 mgl- cKO microglia. N = 3 animals. (F) Representative image of IBA1, ARG1, and Aβ staining. Arg1+ area in microglia was quantified and normalized to the mean of APP/PS1. N = 5 animals. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Functional Assay, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Illustration of Cx43 structure highlighting the 266-283 region. TAT-Cx43 266-283 is the mimetic peptide of this region conjugated to a cell-penetrating peptide TAT. (B) Dye uptake experiment was performed on primary microglia in combination with TAT-Cx43 266-283 treatment at different concentrations. The addition of 1.8 mM Ca 2+ which inhibits hemichannel opening serves as negative control. Scale bar, 10 μm. EtBr signal was quantified, and a non-linear regression analysis was performed to determine the IC50 value. N = 3 coverslips. (C) 50 μM TAT-Cx43 266-283 peptide was added to acute brain slices for 15 min before the EtBr uptake experiment. Followed by IBA1 counter-immunostaining. EtBr signal intensity in IBA1+ microglia was quantified and normalized to TAT-treated APP/PS1 control. N = 4 experiments. (D) Schematic diagram of TAT-Cx43@LNP construction. (E) Electronic microscopic of lipid nanoparticles with different compositions. (F) Measurement of the size of lipid nanoparticles. (G) Measurement of zeta potential of lipid nanoparticles. (H) The size of lipid nanoparticles at different days post-production. (I) Measurement of the peptide release rate at 37 □ of lipid nanoparticles in vitro . (J) Cy5.5-labeled TAT-Cx43 266-283 peptide packaged with DOTAP nanolipid gel (DOTAP-NLG) was i.p. injected into mice, followed by biofluorescence imaging at different time points. (K) representative images of biofluorescence imaging at days 0, 1, and 7 post-injection, revealing the retainment of peptides in the brain. (L) Microplate reader analysis of Cy5.5 intensity of brain lysate and serum collected at day 0, 1, 3, 7, 14 post-injection of TAT-Cx43 peptide alone or with DOTAP-NLG packaging. N = 3 (peptide-alone) or 5 (with packaging) experiments. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Negative Control, Immunostaining, Control, Zeta Potential Analyzer, In Vitro, Labeling, Injection, Imaging

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Primary microglia were treated with 50 μM TAT or TAT-Cx43 266-283 peptide and with or without 1 μM Aβ 25-35 for 3 days before collection for immunostaining. Images show staining of ARG1, CD11b, and DAPI. Scale bar, 5 μm. ARG1 intensity was quantified and normalized to TAT-treated control. (B) Representative image of iNOS, CD11b, and DAPI staining of primary microglia. Scale bar, 5 μm. iNOS intensity was quantified and normalized to TAT-treated control. (C) Dye uptake experiment was performed on APP/PS1 acute brain slices and counterstained with GFAP. EtBr intensity of GFAP+ astrocytes was quantified. N = 4 experiments. (D) gap-FRAP experiment using SR101 dye on acute brain slices from APP/PS1 mice to examine the astrocytic gap junction function. Scale bar, 10 μm. (E) Tissue lysates were collected 1 day after TAT-Cx43 266-283 peptide or DOTAP-NLG packaged TAT-Cx43 266-283 peptide injection, and subjected to microplate reader analysis to analyze the tissue distribution of peptides. (F) Mice were weighed once a week during prolonged TAT@LNP or TAT-Cx43@LNP treatment. (G) Representative electrocardiogram of mice after 6-week treatment. (H) Quantification of Heart rate and electrocardiogram parameters before and after treatment. (I) Heatmap showing serum biochemistry parameters measured before and after TAT@LNP or TAT-Cx43@LNP treatments. Available reference ranges are listed, accompanied by the association between parameters and vital organs. ALB, albumin. TP, total protein. GLB, globulin. TBIL, total bilirubin. GGT, γ-glutamyl transpeptidase. AST, aspartate aminotransferase. ALT, alanine aminotransferase. ALP, alkaline phosphatase. TBA, total bile acids. AMS, serum amylase. LPS, lipase. LDH, lactate dehydrogenase. CK, creatine kinase. Cr, creatinine. UA, uric acid. BUN, blood urea nitrogen. GLU, glucose. TC, total cholesterol. TG, triglyceride. CO2, carbon dioxide. Ca, Calcium. P, phosphate. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Immunostaining, Staining, Control, Injection

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) 8-month-old WT and APP/PS1 mice were treated with TAT-Cx43@LNP or with TAT@LNP as control weekly for 6 weeks, before being subjected to behavioral tests and further histology analysis. (B) Barnes’ maze test was used to analyze mouse cognitive function, specifically, the spatial learning and memory function. The total latency to the target hole, primary latency, and primary exploratory error during the acquisition phase were quantified. (C) The time spent in the target quadrant and the total distance during the probe trial. For WT TAT, WT TAT-CX43, APP/PS1 TAT, and APP/PS1 TAT-CX43, N = 9, 9, 13, 13 animals. (D) Novel object recognition test was used to examine the cognitive function. The discrimination index and the total object exploration time were quantified. (E) Open field test was performed to examine the anxiety traits and locomotor function. Distance and time spent in the center zone and the total distance were quantified. (F) Dye uptake experiment was performed on acute brain slices followed by IBA1 counterstaining. EtBr signal intensity in IBA1+ microglia was quantified and normalized to TAT-treated APP/PS1 control. Scale bar, 50 μm. N = 5 animals. (G) D-serine level in the supernatant of acute brain slices was measured and normalized to the TAT@LNP treated APP/PS1 group. N = 5 animals. (H) Proinflammatory cytokine IL1β and TNFα levels in acute brain slices measured by ELISA. N = 5 animals. (I) Representative images of IBA1 and Methoxy X04 staining. Scale bar, 50 μm. The number of microglia in hippocampus and the periplaque microglia number were quantified. N = 5 animals. (J) Representative images of Methoxy X04 staining in the hippocampus. Scale bar, 50 μm. The number of Aβ plaques number and the percentage of compact plaques were quantified. N = 5 animals. (K) Representative images of RTN3 and Aβ staining in the hippocampus. Scale bar, 10 μm. RTN3+ puncta number at different distances from plaque was quantified. N = 45 (TAT) and 42 (TAT-Cx43) plaques. (L) MitoSox Red staining assay was performed on acute brain slices to analyze hippocampal CA1 neuronal oxidative stress. Scale bar, 50 μm. MitoSox intensity was quantified and normalized to the WT control. N = 5 animals. (M) Theta burst stimulation-induced long-term potentiation (TBS-LTP) was measured on acute brain slices to examine hippocampal synaptic plasticity. (N) Representative field excitatory post synaptic potential (fEPSP) recording before and after LTP induction. (O) Quantification of fEPSP slope during LTP recording experiment. (P) Normalized fEPSP slope after LTP induction. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Comparison of novel object recognition index between untreated animals and TAT@LNP treated animals. (B) Quantification of total distance, and primary distance to the target hole during Barnes’ maze acquisition trial. (C) Quantification of distance spent in the target quadrant during Barnes’ maze probe trial. (D) Quantification of immobility time, and standing time during the open field test. (E) Representative image of Cx43 staining of hippocampus and quantification of Cx43 intensity. Scale bar: 100 μm. N = 5 animals. (F) 3D rendering of IBA1 staining and Cx43 signal within IBA1+ domain. Scale bar: 10 μm. The microglial Cx43 signal was quantified, N = 5 animals. (G) Dye uptake experiment performed on acute brain slice followed by GFAP staining, Scale bar: 20 μm. The EtBr intensity of GFAP+ astrocyte was quantified. (H) Representative image of gap-FRAP experiments and quantification of SR101 dye intensity. Scale bar: 10 μm. (I) Representative image of IBA1, ARG1, and Aβ staining. Scale bar: 10 μm. Arg1+ area in microglia was quantified and normalized to the mean of APP/PS1. N = 5 animals. (J) Quantification of periplaque microglia and distal microglia normalized to the area. N = 5 animals. (K) the number of periplaque microglia was plotted against the plaque perimeter. The linear regression analysis was performed. (L) 3D rendering of Aβ plaque and Aβ plaque within IBA1+ microglial domain. Scale bar: 10 μm. The fraction of Aβ plaque within IBA1+ microglial domain was quantified. N = 5 animals. (M) Sholl analysis of periplaque and distal microglia. (N) Quantification of Methoxy X04+ Aβ plaque area, plaque core area ratio, and plaque circularity. N = 5 animals. (O) Representative image of MBP staining of hippocampal CA1 region. Scale bar: 10 μm. MBP+ area in the hippocampus was quantified. N = 5 animals. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Comparison, Staining, Slice Preparation

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) 3.5-month-old WT and APP/PS1 mice were treated with TAT-Cx43@LNP or with TAT@LNP as control weekly for 8 weeks, before being subjected to histology analysis. (B) MitoSox Red staining assay was performed on acute brain slices to analyze hippocampal CA1 neuronal oxidative stress. Scale bar, 50 μm. MitoSox intensity was quantified and normalized to the WT control. N = 5 animals. (C) Representative images of RTN3 and Aβ staining in the hippocampus. Scale bar, 10 μm. RTN3+ puncta number at different distances from plaque was quantified. N = 26 (TAT) and 29 (TAT-Cx43) plaques. (D) Representative images of Aβ and postsynaptic element labelling by Homer1. Scale bar: 10 μm. Homer1+ puncta intensity at different distances from plaque was quantified. N = 11 (TAT) and 13 (TAT-Cx43) plaques. (E) Representative images of IBA1 and Methoxy X04 staining. Scale bar, 50 μm. (F) Quantification of Aβ plaque number. N = 5 animals. (G) Higher resolution images of IBA1 and Methoxy X04 staining. Scale bar, 10 μm. (H) Quantification of the percentage of compact plaque. N = 5 animals. (I) For each plaque, the number of periplaque microglia was quantified, and normalized to the plaque perimeter. N = 5 animals. (J) 3D reconstruction image of the panel (G) displaying Aβ plaque and the fraction of Aβ plaque within IBA1+ microglia domain. (K) The ratio between plaque within microglia and total plaque volume was quantified. N = 5 animals. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Turning microglia neuroprotective: Towards connexin43-specific therapy of Alzheimer’s disease

doi: 10.1101/2024.08.06.606883

Figure Lengend Snippet: (A) Dye uptake experiment was performed on APP/PS1 acute brain slices and counterstained with IBA1. Scale bar: 10 μm. EtBr intensity of IBA1+ microglia was quantified and normalized to WT. N = 5 experiments. (B) Quantification of the number of total microglia, periplaque microglia, and distal microglia normalized to the area. N = 5 animals. (C) Quantification of Plaque perimeters. N = 5 animals. (D) Sholl analysis of periplaque and distal microglia in APP/PS1 mice as well as WT microglia under TAT or TAT-CX43@LNP treatment. For periplaque microglia, N = 65 (TAT) and 72 (TAT-CX43) cells. For distal microglia, N = 50 (TAT) and 62 (TAT-CX43) cells. For WT, N = 42 (TAT) and 45 (TAT-CX43) cells. Dot plots show mean ± SEM, each data point, and p -value. Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies used are Rabbit-anti-Cx43 (Merck, C6219), Rabbit-anti-PANX1 (Thermo, 487900), Rabbit-anti-Cx30 (Thermo, 712200), Goat-anti-IBA1 (Abcam, ab5076), Goat-anti-GFAP (Abcam, ab53554), Rat-anti-GFAP (Abcam, ab279291), Rabbit-anti-ARG1 (Thermo, PA529645), Mouse-anti-Aβ 6E10 (Biolegend, 803001), Rat-anti-MBP (Millipore, MAB386), Rabbit-anti-RTN3 (Millipore, ABN1723), Lamp1 (Biolegend, 121602).

Techniques:

Journal: Toxicological Sciences

Article Title: 3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa

doi: 10.1093/toxsci/kfab153

Figure Lengend Snippet: Human KGN cells formed stable 3D spheroids that expressed connexin 43 and CYP19 aromatase enzyme. Representative image of formalin-fixed, paraffin-embedded sections of KGN spheroids, stained with hematoxylin and eosin (A, B), immunostained for Cx43 (green) (C) or CYP19 enzyme (green) (D), isotype control for both anti-Cx43 and anti-CYP19 antibody (green) (C, D), cell membrane with wheat germ agglutin (WGA, red) (C), and nuclei with DAPI (blue) (C, D). KGN spheroids were fixed at 24 h postself-assembly (A, C, and D), or at 72 h after incubation in media containing 10−7M testosterone (B). Figure 2A includes an enlarged inset of a spheroid section with black arrows pointing at the pyknotic nuclei. Scale bar = 50 µm. Three independent experiments, n = 2–3 gels per experiment, each with 256 spheroids.

Article Snippet: Slides stained with anti-Cx43 were rinsed 3 times in TBS, followed by 3 rinses with Hank’s balanced salt solution (HBSS) without phenol red (Fisher Scientific, MT21023CV), then was first counterstained with wheat germ agglutinin Texas Red-X conjugate (Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"W21405","term_id":"1297900","term_text":"W21405"}} W21405 ; 5 μg/ml) in HBSS for 10 min to reveal cell membrane, followed by 3 rinses with HBSS, and then counterstained with DAPI Solution for 10 min and mounted using Prolong Diamond Antifade Mountant.

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Incubation