Journal: IBRO neuroscience reports

Article Title: IL-33 relieves nerve injury by mediating microglial polarization in neuromyelitis optica spectrum disorders via the IL-33/ST2 pathway.

doi: 10.1016/j.ibneur.2024.07.008

Figure Lengend Snippet: Fig. 5. Microglial cells were activated and transformed to the M2 phenotype after treatment with IL-33. (a) Western blot images and the quantification of CD206 and CD40 levels after treatment with IL-33 or NMOD serum. (b) BV2 cells were labeled with Iba-1 and CD68 for immunofluorescence. (c) Expression levels of PSD95 were observed by immunofluorescence after treatment with IL-33 or NMOSD serum. One-way analysis of variance was used for statistical analyses. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: For protein immunoreactivity in cells, the primary cortical neurons or BV2 cells were fixed with 4 % paraformaldehyde for 30 min at RT and treated with 0.5 % Triton for 10 min. After being washed with PBS, the cells were blocked with 5 % bovine serum albumin (BSA) for 1 h and then incubated with a rabbit antiPSD95 antibody (1:100 dilution, PA2295, Boster), anti-CD68 antibody (1:200 dilution, 28058–1-AP, Proteintech), anti-IBA1 antibody (1:100 dilution, CY7217, Abways), or anti-ST2 antibody (1:200 dilution, PRS3363, Sigma) at 4 ◦C overnight.

Techniques: Transformation Assay, Western Blot, Labeling, Immunofluorescence, Expressing

Journal: Stem cell research & therapy

Article Title: Subretinal microglia support donor photoreceptor survival in rd1 mice.

doi: 10.1186/s13287-024-04052-0

Figure Lengend Snippet: Fig. 2 Macrophage and microglia detection after transplantation. A. In rd1 mice, macrophages (CD68+) were observed in the choroid when donor cells either survived (left) or died (middle). In wild-type mice, dead donor cells were engulfed by macrophages (right). Scale bar = 10 μm. B. In trans-scleral injection group, while microglial cells surrounded surviving donor cells in rd1 mice (i), they could hardly be detected around dead cell debris (ii). In wild-type mice, microglia/macrophages engulfed dead donor cells (iii). Following trans-vitreous injection, both dead (arrows) and surviving donor cells (arrowheads) were found in the subretinal space. While macrophages/microglia engulfed the dead cells (arrows), microglia were not close to surviving cells (iv). C. A Comparison of chemokines expression between wild-type and rd1 mice showed a significant increase in the mRNA levels of ccl2, ccl3 and cxcl2 in rd1 mice. D. The comparison of cytokine levels between wild-type and rd1 mice showed no significant difference. E. No significant differences were found in oxidative stress factors between wild-type and rd1 mice. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following primary antibodies were used in the study: CD68 (1:400,#97778, Cell Signaling Technology), Iba1 (1:200, #019-19741, Wako), Galectin-3 (1:200, #sc-53127, Santa Cruz), F4/80(1:400, #71299, Cell Signaling Technology), CD11b (1:200, 101,209, Biolegend).

Techniques: Transplantation Assay, Injection, Comparison, Expressing

Journal: bioRxiv

Article Title: Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

doi: 10.1101/2021.12.22.473777

Figure Lengend Snippet: (A) Schematic of LV.hexa+LV.hexb injection and immunofluorescence (IF) confocal image showing clearance of GM2 storage and reduction in CD68+ in a region of SD brain tissue (box) close to the LV injection site. Upper panel: GM2, red; lower panel: CD68, red; IBA1, blue; nuclei are counterstained with Hoechst (white). Scale bar, 25 μm. (B-C) Percentages of donor-derived GFP+ cells in the peripheral blood (PB; B) and bone marrow (BM; C) of BMT- and IC GT+BMT-treated SD and WT mice evaluated at 30 d (PB) and 60 d (BM) after BMT. The percentage of GFP+ cells in the BM of tgGFP mice (donors) is shown for comparison. (D) Cellular composition of the BM analyzed 60 d after transplantation into BMT- and IC GT+BMT-treated SD mice and age-matched UT controls (WT, SD, and tgGFP mice). CD19, marker for B cells; CD11b, marker for monocytes, neutrophils, and NK cells; CD3, marker of mature T cells. Data represent the mean ± SEM; n= 3-13 mice/group. (E) IF image showing distribution of GFP+ cells in the whole brain of IC GT+BMT-treated SD mouse at 120 d. Boxes highlighting brain regions in (F) at higher magnification. (F) IF pictures showing GFP+ cells engrafted into olfactory bulb (OB), hippocampus (Hip), thalamus (Thal), and Pons/medulla (Pons) of IC GT+BMT-treated SD mice at 120 d and 240 d. In E and F: direct GFP fluorescence, green; nuclei counterstained with Hoechst, grey. Scale bars, 3.000 μm (E) and 500 μm (F).

Article Snippet: The following primary antibodies were employed: chicken anti-GFP (1:500; ab-13970, Abcam, Cambridge, UK); glial fibrillary acidic protein (GFAP; polyclonal 1:1000, ZO334, DAKO-Agilent, Santa Clara, CA, USA; monoclonal 1:1000, MAB 3402, Millipore, Burlington, Massachusetts, United States); rabbit anti Iba1 (1:100; 019-19741, Wako Chemicals, Neuss, Germany); rat anti CD68 (1:200; clone FA-11/MCA1957, Bio-Rad Laboratories Inc, Hercules, CA, USA); rabbit anti LAMP1 (1:200, AB24170; Abcam, Cambridge, UK); rat anti LAMP1 hybridoma (1:300; 1D4B, DSHB at The University of Iowa, USA); mouse anti GM2 (1:500 A2576; TCI EUROPE); rabbit anti Calbindin (1:1000, CB-38A Swant, Burgdorf, Switzerland).

Techniques: Injection, Immunofluorescence, Derivative Assay, Comparison, Transplantation Assay, Marker, Fluorescence

Journal: bioRxiv

Article Title: Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

doi: 10.1101/2021.12.22.473777

Figure Lengend Snippet: (A) Enzymatic activity measured in the TEL, CB, and SC tissues of IC GT-, BMT-, and IC GT+BMT-treated SD mice and age-matched untreated (UT) SD mice at 120 d and 240 d. Enzymatic activity was measured as the degradation of the artificial substrates MUG (left graph) and MUGS (right graph) and expressed as percentage of age-matched UT WT control data. (B) Enzymatic activity (MUG, MUGS; expressed as nmol/mg/h) in the TEL, CB, and SC tissues of WT and Het mice at 120 d and 240 d. * p < 0.01, ** p < 0.005, *** p < 0.001, **** p < 0.0001. (C-D) Enzymatic activity (MUG, MUGS; expressed as percentage of age-matched UT WT controls) measured in the cerebrospinal fluid (CSF; C) and bone marrow (BM; D) of treated SD mice (IC GT, BMT, and IC GT+BMT) and UT controls (Het, SD) at 120 d and 240 d. * p < 0.05 (C); **** p < 0.0001, *** p < 0.001 SD IC GT 120 d vs all other treatments (D). Combined treatment vs WT UT, not significant. (E-F) Confocal IF images showing donor-derived GFP+ cells (green) expressing macrophagic marker Iba1 (red) and CD68 (blue) in liver (E) and spleen (F) of BMT-treated SD mice at 120 d. Scale bar, 50 μm. (G-H-I) Enzymatic activity measured in liver (G), spleen (H), and sciatic nerve (I) of treated SD mice (IC GT, BMT, and IC GT+BMT) and UT controls (Het, SD) at 120 d and 240 d. Enzymatic activity was measured as the degradation of MUG and MUGS and expressed as percentage of age-matched UT WT controls. ** p < 0.01, **** p < 0.0001 SD IC GT 120 d vs all other treatments (G-H). **** p < 0.0001, ** p < 0.01 SD IC GT+BMT 240d vs all other treatments (I). Combined treatment vs Het (liver) and WT (spleen), p>0.05. Data in A-D and G-I are expressed as the mean ± SEM; n = 3-9 mice/group. Data analyzed by 2-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The following primary antibodies were employed: chicken anti-GFP (1:500; ab-13970, Abcam, Cambridge, UK); glial fibrillary acidic protein (GFAP; polyclonal 1:1000, ZO334, DAKO-Agilent, Santa Clara, CA, USA; monoclonal 1:1000, MAB 3402, Millipore, Burlington, Massachusetts, United States); rabbit anti Iba1 (1:100; 019-19741, Wako Chemicals, Neuss, Germany); rat anti CD68 (1:200; clone FA-11/MCA1957, Bio-Rad Laboratories Inc, Hercules, CA, USA); rabbit anti LAMP1 (1:200, AB24170; Abcam, Cambridge, UK); rat anti LAMP1 hybridoma (1:300; 1D4B, DSHB at The University of Iowa, USA); mouse anti GM2 (1:500 A2576; TCI EUROPE); rabbit anti Calbindin (1:1000, CB-38A Swant, Burgdorf, Switzerland).

Techniques: Activity Assay, Control, Derivative Assay, Expressing, Marker, Comparison

Journal: bioRxiv

Article Title: Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis

doi: 10.1101/2021.12.22.473777

Figure Lengend Snippet: (A) Relative mRNA expression of neuroinflammatory cytokines (CCL3, CCL5, macrophage (CD68), and astrocytic (GFAP) markers in whole brain lysate (TEL, CB) of treated mice (IC GT, BMT, and IC GT+BMT) at 120 d and 240 d and age-matched untreated controls (WT, Het, and SD). Data are expressed as fold-change with respect to WT (set as 1) after normalization to Gapdh expression. (B) Representative western blot and relative quantification showing the expression of GFAP protein in treated mice (IC GT, BMT, and IC GT+BMT) at 120 d and 240 d and age-matched untreated controls (WT, Het, and SD). Data are expressed as fold-change to WT (set as 1) after normalization to calnexin (CNX) expression. Data in A and B represent the mean ± SEM; n = 3-11 animals/group. One-way ANOVA followed by Kruskal Wallis multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) 3D projections of Z-stack images showing the presence of donor-derived GFP+ cells (green) and resident cells expressing Iba1 (microglia, red) and CD68 (macrophages, blue) in the TEL of IC GT+BMT-treated SD mice at 120 d and 240 d and UT controls (WT, SD). Nuclei counterstained with Hoechst, grey. Images were acquired at 40X magnification.

Article Snippet: The following primary antibodies were employed: chicken anti-GFP (1:500; ab-13970, Abcam, Cambridge, UK); glial fibrillary acidic protein (GFAP; polyclonal 1:1000, ZO334, DAKO-Agilent, Santa Clara, CA, USA; monoclonal 1:1000, MAB 3402, Millipore, Burlington, Massachusetts, United States); rabbit anti Iba1 (1:100; 019-19741, Wako Chemicals, Neuss, Germany); rat anti CD68 (1:200; clone FA-11/MCA1957, Bio-Rad Laboratories Inc, Hercules, CA, USA); rabbit anti LAMP1 (1:200, AB24170; Abcam, Cambridge, UK); rat anti LAMP1 hybridoma (1:300; 1D4B, DSHB at The University of Iowa, USA); mouse anti GM2 (1:500 A2576; TCI EUROPE); rabbit anti Calbindin (1:1000, CB-38A Swant, Burgdorf, Switzerland).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To examine macrophage activation and Purkinje cells loss in the cerebellum, the location and protein levels of CD 68 (macrophage marker) and Calbindin (Purkinje cell marker) were examined in the cerebella of mice at different time points. Half of cerebella of mice were fixed for immunofluorescence staining, other half of the cerebella were fresh frozen for western blot analysis. (A) Images representatives of anti-Calbindin and anti-CD68 at the ages of 1,3, and 6 months. (B) Quantitative analysis of positive cells number of immunofluorescence staining signal of CD68. (C) Quantitative analysis of positive cells number of immunofluorescence staining signal of Calbindin. (D) Western blot images of CD68 and Calbindin. (E) Quantitative analysis of western blot of CD68 and Calbindin. Note: Data was presented as mean ± SEM and unpaired t-test or one-way ANOVA Turkey multiple comparison test was used for quantitative analysis in GraphPad Prism 10. * p <0.05, **** p <0.0001.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Activation Assay, Marker, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To examine EGFR expression in macrophage, the colocalization of CD68 and EGFR were investigated in the cerebella of mice at different time points. Images representatives of anti-CD68 and anti-EGFR at the ages of 1,3, and 6 months.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: To assess the expression of phosphorylated (Tyr1068) EGFR (pEGFR) in macrophage, the location and protein levels of pEGFR and CD68 were examined in the cerebella of mice at different time points. Half of cerebella of mice were fixed for immunofluorescence staining, other half of the cerebella were fresh frozen for western blot analysis. (A) Images representatives of anti- pEGFR and anti-CD68 at the ages of 1,3, and 6 months. (B) Quantitative analysis of positive cells number of immunofluorescence staining signal of pEGFR (D) Western blot images of pEGFR. (E) Quantitative analysis of western blot of pEGFR. Note: Data was presented as mean ± SEM and unpaired t-test or one-way ANOVA Turkey multiple comparison test was used for quantitative analysis in GraphPad Prism 10. * p <0.05, **** p <0.0001.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Association of cerebellar inflammation and neurodegeneration in a novel spinocerebellar ataxia type 13 mouse model

doi: 10.1101/2024.10.28.620701

Figure Lengend Snippet: Pearson correlation was analyzed among EGFR/pEGFR, CD68, GFAP, Iba-1 and Calbindin according to our quantitative expression data. (A) Correlation between EGFR positive cell number and Calbindin positive cell number. (B) Correlation between pEGFR positive cell number and Calbindin positive cell number. (C) Correlation between CD68 positive cell number and Calbindin positive cell number. (D) Correlation between EGFR positive cell number and CD68 positive cell number. (E) Correlation between pEGFR positive cell number and CD68 positive cell number. (F) Correlation between pEGFR positive cell number and the intensity of GFAP positive signal.

Article Snippet: In the last step slides were incubated overnight the following conjugated antibodies: anti-Calbindin 488 (Cell signaling, #13176, 1:100), anti-Glial Fibrillary Acidic Protein (GFAP) Alexa Fluor 488 (Invitrogen, A-21294, 1:200), Ionized calcium binding adaptor molecule 1 (Iba1) Alexa Fluor 488 (Cell Signaling, #20825, 1:100), and CD68 Alexa Fluor 488 (Cell Signaling, #51644, 1:200).

Techniques: Expressing

Journal: bioRxiv

Article Title: Butyrate Rescues Cardiac Metabolic Dysfunction in Hypertensive Heart Failure with Preserved Ejection Fraction

doi: 10.1101/2025.11.28.690152

Figure Lengend Snippet: (a) Body weight was significantly different between WT and dTGR (n= 14 WT+NaCl, n=15 dTGR+NaCl, n=10 dTGR+butyrate). (b) cardiac hypertrophy index (heart weight to tibia length ratio) (n= 14 WT+NaCl, n=10 dTGR+NaCl, n=9 dTGR+butyrate. (c) Quantification of cardiomyocyte cross-sectional perimeter from WGA staining (left) (n= 10 WT+NaCl, n=8 dTGR+NaCl, n=10 dTGR+butyrate) and representative images (right, scalebar 20µm). Relative expression of (d) Anp, (e) Bnp, f) Timp, and (g) Opn to 36b4 measured by RT-qPCR (n= 13-12 WT+NaCl, n=8-6 dTGR+NaCl, n=10-8 dTGR+butyrate). (h) Quantification of fibrotic area from sirius red histologic staining (left) (n= 9 WT+NaCl, n=7 dTGR+NaCl, n=10 dTGR+butyrate), and representative images (right, scalebar 20µm). (i) Quantification of ED1-positive cells per field of view in ED1 (CD68) immune-histological staining (left) (n= 10 WT+NaCl, n=8 dTGR+NaCl, n=10 dTGR+butyrate), and representative images (right, scalebar 50µm. Outliers were removed upon statistical testing. Data are presented as boxplots (IQR) with whiskers min to max. (a-i) . *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (a) Kruskal-Wallis test with Dunn’s multiple comparison, (b-i) ordinary one-way ANOVA with Dunnett’s multiple comparison.

Article Snippet: Primary antibody (Rabbit anti-rat Fibronectin antibody [1:75] Ab23751; Abcam, Mouse anti-rat CD68 antibody | Clone ED1 [1:50] MCA341R; Bio-Rad) or biotinylated wheat germ agglutinin (WGA [1:100] B1025 VectorLab) or sirius red solution (0.1% in picric acid, Sigma) was diluted in 10% NDS and applied to the slice.

Techniques: Staining, Expressing, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

doi: 10.1101/216531

Figure Lengend Snippet: (A) Representative images of the lateral ventricle of CTRL ASO mice stained with CD68 (green), GFAP (red) and DAPI (blue). CD11b cells were positively stained in the lateral ventricle and negatively stained for TMEM119. (B) Representative stitched images of the lateral ventricle in bregma −2.3mm (approximately) stained with CD68 (green) and GFAP (red) between CTRL ASO and C1INH ASO treated mice. (C) Representative images zooming x10 in the lateral ventricle shown in B, showing migrating CD68 positive cells in the lateral ventricle wall area in C1INH ASO treated mice brains. (D) Quantification of CD68 by intensity of staining in CTRL ASO vs. C1INH ASO treated mice brains (100±20.19 vs. 43.94±10.45, p=0.025, n=9). (bar scales are 100μm scale)

Article Snippet: The primary antibodies used were: anti-GFAP (DAKO Z0334); anti-CD11b (Abcam ab-8878); anti-TMEM (Abcam ab209064); anti-PECAM1 (BD Pharmingen 550274); anti-CD68 (AbD Serotec MCA1957GA); anti-CD206 (Thermo PA5-46994); anti-Fibrinogen (Dako A0080); IgG (Thermo scientific); anti-laminin (Fisher scientific RT-795-PO); and anti-iNOS (Abcam ab-129372).

Techniques: Staining

Journal: bioRxiv

Article Title: Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

doi: 10.1101/216531

Figure Lengend Snippet: Representative images of GFAP (red) staining and CD68 (green) in the neurovascular units.

Article Snippet: The primary antibodies used were: anti-GFAP (DAKO Z0334); anti-CD11b (Abcam ab-8878); anti-TMEM (Abcam ab209064); anti-PECAM1 (BD Pharmingen 550274); anti-CD68 (AbD Serotec MCA1957GA); anti-CD206 (Thermo PA5-46994); anti-Fibrinogen (Dako A0080); IgG (Thermo scientific); anti-laminin (Fisher scientific RT-795-PO); and anti-iNOS (Abcam ab-129372).

Techniques: Staining

Journal: bioRxiv

Article Title: Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

doi: 10.1101/216531

Figure Lengend Snippet: Co-localization of CD68 positive cells and CD206.

Article Snippet: The primary antibodies used were: anti-GFAP (DAKO Z0334); anti-CD11b (Abcam ab-8878); anti-TMEM (Abcam ab209064); anti-PECAM1 (BD Pharmingen 550274); anti-CD68 (AbD Serotec MCA1957GA); anti-CD206 (Thermo PA5-46994); anti-Fibrinogen (Dako A0080); IgG (Thermo scientific); anti-laminin (Fisher scientific RT-795-PO); and anti-iNOS (Abcam ab-129372).

Techniques:

Journal: bioRxiv

Article Title: Knockdown of endogenous circulating C1 inhibitor induces neurovascular impairment, neuroinflammation and cognitive decline

doi: 10.1101/216531

Figure Lengend Snippet: (A) Representative image of the ventricular wall stained with CD68 (green), CD206 (red) and DAPI (blue) showing infiltrating blood derived cells that are not co-localized with CD206 (white arrows) (B) Representative image of positive CD68 cells that are not co-localizing with CD206 in the brain parenchyma. (C) Representative image from bregma −2.54mm of C1INH ASO treated mice brain showing migrating CD68 positive cells in the optic tract region. (D) TMEM119 (red) was negatively stained whereas CD11b (green) was positively stained in the optic tract of C1INH ASO treated mice brain (bar scales are 100μm scale).

Article Snippet: The primary antibodies used were: anti-GFAP (DAKO Z0334); anti-CD11b (Abcam ab-8878); anti-TMEM (Abcam ab209064); anti-PECAM1 (BD Pharmingen 550274); anti-CD68 (AbD Serotec MCA1957GA); anti-CD206 (Thermo PA5-46994); anti-Fibrinogen (Dako A0080); IgG (Thermo scientific); anti-laminin (Fisher scientific RT-795-PO); and anti-iNOS (Abcam ab-129372).

Techniques: Staining, Derivative Assay