Journal: PLoS ONE

Article Title: Cholesterol Diet Withdrawal Leads to an Initial Plaque Instability and Subsequent Regression of Accelerated Iliac Artery Atherosclerosis in Rabbits

doi: 10.1371/journal.pone.0077037

Figure Lengend Snippet: Panel (A) Representative images of hematoxylin and eosin stained sections of all groups (Scale bar = 500 µm). Panel (B) Representative images of Oil Red O stained cryostat sections of all groups (Scale bar = 50 µm). Panel (C) Immunohistochemical staining showing CD68 positive cells in respective groups. (Scale bar = 50 µm) (D) Quantitative analysis of percentage lipid area (lumen area included) in respective groups (E) Quantitative analysis of CD68 positive labelling in respective groups. **p<0.01 and ***p<0.001 vs normal; ## p<0.01 and ### p<0.001 vs Baseline. (* indicates lumen, arrow heads indicate positive staining)

Article Snippet: Paraffin-embedded consecutive sections were stained with the following antibodies: Mouse monoclonal anti rabbit CD68 (RAM11, Dako, USA), alpha smooth muscle actin (1A4, Sigma, USA) and MMP-9 (56-2A4, Calbiochem, USA).

Techniques: Staining, Immunohistochemical staining

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Regnase-3 –deficient mice develop hypertrophic lymph nodes. (A) Frequency of mice showing lymphadenopathy in a cohort of 24 Regnase-3 −/− mice and 24 Regnase-3 +/+ littermate controls at 3–6.5 mo of age. (B) Photography of skin-draining lymph nodes of four Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 5 mo of age. (C) Representative photography of inguinal lymph nodes of a Regnase-3 −/− mouse at 5 mo of age. Arrow indicates hypertrophic lymph node. (D) H&E staining and immunohistochemical analysis of B cells (B220), T cells (CD3), and macrophages (F4/80) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 3/3). Magnification of images is indicated in brackets. Bars, 1,000 µm. (E) Immunohistochemical analysis of macrophages (CD68) in skin-draining lymph nodes of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images from n = 6/6). Images of enlarged and small lymph nodes are taken from the identical Regnase-3 −/− mouse. Top right: Frequency of strong positive (pos.) pixels in CD68 immunohistochemical sections of the lymph nodes was determined by Definiens software ( n = 6/6). Bars, 500 µm. (F) Top: Frequencies of B cells (CD19 + ) and T cells (CD90 + ) in enlarged and normal-sized lymph nodes of the same Regnase-3 −/− mouse and its Regnase-3 +/+ littermate control at 6 mo of age, assessed by flow cytometry (representative blots of n = 6/6). Number of total cells in lymph nodes of Regnase-3 +/+ mice and Regnase-3 −/− littermates ( n = 6/6). Bottom: Frequencies of B cells (CD19 + ), T cells (CD90 + ), CD4 + and CD8 + T cells, and CD11b + cells in enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermate controls at 6 mo of age, assessed by flow cytometry ( n = 6/6). Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Staining, Immunohistochemical staining, Software, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3

doi: 10.1084/jem.20181762

Figure Lengend Snippet: Systemic IFN signaling in Regnase-3 −/− mice. (A) CD19 + B cells were isolated from enlarged lymph nodes of Regnase-3 −/− mice and their Regnase-3 +/+ littermates ( n = 3/3), and RNA was isolated and subjected to RNA sequencing. Heatmap of RNA sequencing data for all significantly up-regulated (≥2 log2 fold) genes in Regnase-3 −/− B cells is shown. GO term association to “response to IFNβ” and “response to IFNγ” is indicated for each gene. (B) Serum cytokines in Regnase-3 −/− mice and Regnase-3 +/+ littermates at 6 mo of age ( n = 8/8), assessed by multiplex assay. (C) Stat1 mRNA expression in tissues from Regnase-3 +/+ and Regnase-3 −/− mice at 8 mo of age, assessed by quantitative RT-PCR, normalized to Hprt relative (rel.) to their expression in Regnase-3 +/+ mice ( n = 5/5). (D) Left: Immunohistochemical analysis of MHC-II in liver sections of Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ controls (representative images). Magnification of images is indicated in brackets. Bars, 250 µm. Right: Frequency of strong positive pixels in MHC-II immunohistochemical sections of lung, kidney, and liver were determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). (E) Frequency of MHC-II–positive macrophages (CD68 + ) of all CD68 + macrophages in the lung of Regnase-3 −/− mice with lymphadenopathy and their Regnase-3 +/+ littermate controls, assessed in immunohistochemical, consecutive sections ( n = 5/6). (F) Left: Immunohistochemical analysis of pSTAT1 in skin-draining lymph nodes from Regnase-3 −/− mice with lymphadenopathy and Regnase-3 +/+ littermate controls (representative images). Right: Frequency of strong positive pixels in pSTAT1 immunohistochemical sections of lymph nodes, determined by Definiens software ( n = 6 Regnase-3 −/− mice and 6 Regnase-3 +/+ littermate controls). Bars, 500 µm. Data are represented as mean ± SEM and were compared by Mann–Whitney U test (*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant).

Article Snippet: Staining antibodies were anti-B220 (RA3-6B2, rat-IgG2a; BD), anti-CD3 (SP7, rabbit IgG; Zytomed), anti-F4/80 (BM8, rat-IgG2a; Linaris), anti-MHC-II (M5/114.15.2, rat IgG; Novus Biologicals), anti-CD68 (Ab125212, rabbit IgG; Abcam) anti-ki67 (SP6, rabbit IgG; Thermo Fisher Scientific), and anti-Tyr701-phospho-STAT1 (58D6, rabbit IgG; Cell Signaling).

Techniques: Isolation, RNA Sequencing Assay, Multiplex Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Software, MANN-WHITNEY

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