Journal: Current Issues in Molecular Biology

Article Title: Anti-Inflammatory and Synaptic Protective Effects of TNF-α Inactivation in the MDX Mouse Model

doi: 10.3390/cimb48030270

Figure Lengend Snippet: Anti-GFAP immunohistochemistry of cross-sections of the lumbar enlargement at 20× magnification. ( B , D ) Elevated expression in MDX animals compared to C57BL/10 ( A , C , E , G ). ( F , H ) Positive regulation of expression following treatment at doses of 6 and 12 mg/Kg. Scale bar: 25 μm. ( I ) Graph of GFAP immunohistochemistry in lumbar enlargement cross-sections, with quantification of the integrated density of pixels and statistical significance of intergroup comparisons: ns, non-significant; **** p < 0.0001. Mean ± SEM.

Article Snippet: GFAP , Santa Cruz , rabbit anti-human , 1:750.

Techniques: Immunohistochemistry, Expressing

Journal: Cell reports

Article Title: Angiopoietin-2 aggravates Alzheimer’s disease by promoting blood-brain barrier dysfunction and neuroinflammation

doi: 10.1016/j.celrep.2025.116621

Figure Lengend Snippet: (A and B) Increased CD74 + activated microglia co-labeled with Iba1 in the brain cortex of 6-month-old 5xFAD mice treated with ANGPT2 AAV compared to control-AAV-treated mice (A), with corresponding quantification (B). Arrowheads indicate CD74 + activated microglia. Scale bars: 50 μm. (C and D) Representative images (C) and quantification (D) showing increased cell body diameter and decreased dendritic length of Iba1 + microglia in ANGPT2-AAV-treated 5xFAD mice compared with controls. Asterisks mark the centers of Iba1 + microglial nuclei. Scale bars: 10 μm. (E and F) Elevated GFAP + reactive astrocytes co-labeled with S100β in the cortex of ANGPT2-AAV-treated 5xFAD mice compared with controls (E), with corresponding quantification (F). Arrowheads indicate GFAP + reactive astrocytes. Scale bars: 50 μm. (G and H) Morphological analysis of GFAP + reactive astrocytes showing increased branching and area coverage in ANGPT2-AAV-treated 5xFAD mice compared to control-AAV-treated mice (G), with corresponding quantification (H). Scale bars: 50 μm (white) and 20 μm (yellow). (I) Cytokine profile analysis showing increased levels of pro-inflammatory cytokines in ANGPT2-AAV-treated 5xFAD mice compared to control-AAV-treated mice. Each dot indicates an individual mouse for (B), (F), (H), and (I). Data are presented as the mean ± SEM.

Article Snippet: Next, sections were incubated overnight at 4°C with the following primary antibodies: For mouse tissue sections: CD31 (Thermo Fisher Scientific, MA3105), β-amyloid (Abcam, ab2539, ab126649), TIE2 (Regeneron Pharmaceuticals Inc., human monoclonal, REGN1376), pTIE2 (R&D systems, AF2720), ANGPT2 (Regeneron Pharmaceuticals Inc., human monoclonal, REGN910), PDGFR β (Thermo Fisher Scientific, 14-1402-82), fibrin(ogen) (Dako, A0080), claudin-5 (Thermo Fisher Scientific, 34-1600), GFAP (Aves Lab, GFAP), S100 β (Abcam, ab52642), Iba1 (Fujifilm Wako, 019-19741), CD74 (BioLegend, 151002), occludin (Thermo Fisher Scientific, 71-1500), PLVAP (BD Biosciences, 550563), and caveolin-1 (Santa Cruz Biotechnology, sc53564).

Techniques: Labeling, Control

Journal: Nature neuroscience

Article Title: Therapeutically viable generation of neurons with antisense oligonucleotide suppression of PTB.

doi: 10.1038/s41593-021-00864-y

Figure Lengend Snippet: Fig. 3 | Transition of GFAP:tdTomato+ cells into new neurons results in no depletion in the total GFAP cell number. a, Images of newly induced neurons (iNeurons) expressing NeuN (green) and tdTomato (red) (NeuN visualized by IF and tdTomato by direct fluorescence); blue, DAPI staining for DNA; white, GFAP visualized by IF; the experiment was reproduced four times, independently, with similar results. b, Total number of tdTomato+NeuN− cells (left) or tdTomato+NeuN+ cells (right), either with or without GFAP labeling, per dentate gyrus, 2 months after ICV injection of PTB-ASO. Data are presented as mean ± s.e.m. (left: tdTomato/NeuN: 176.8 ± 34.92; tdTomato/NeuN/GFAP mean: 167 ± 34.32; right: tdTomato/NeuN mean: 28.88 ± 4.87; tdTomato/NeuN/GFAP mean: 0.87 ± 0.12; n = 4 biological repeats; two-tailed t-test **P = 0.0012). c, Representative images of human organoids 1 month after PTB-ASO or control-ASO application in the growth media GFAP (green) and Nestin (red) (both visualized by IF); blue, DAPI staining for DNA; the experiment was reproduced three times, independently, with similar results. d, Representative images of a dentate gyrus taken 2 months after ICV injection of control or PTB-ASO2 into 3-month-old and 1-year-old mice; white, GFAP visualized by IF; the experiment was reproduced three times for control and aged mice and four times for 5-month-old mice, independently, with similar results. e, Total GFAP-positive cells per dentate gyrus area of 5-month- and 1.2-year-old PTB-ASO- or control-injected mice. Data are presented as mean ± s.e.m. (control mean: 1,074 ± 83.55; 5 months PTB-ASO mean: 1,006 ± 82.45; 1.2 year-old PTB-ASO mean: 1,214 ± 104.8; n = 3, n = 4, n = 3 biological repeats).

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Rabbit-PTBP1 (Abclonal; Cat# - A6107; IF concentration - 1:1000); Mouse-GFAP (Dako; Cat# - M0761; IF concentration - 1:500); Mouse-NeuN (Millipore; Cat# - MAB377; IF concentration - 1:100); Rabbit-pan-ASO (Ionis pharmaceutical, homemade antibody; Cat# -13545; IF concentration - 1:200); Chicken-MAP2 (Abcam; Cat# - ab5392; IF concentration - 1:1000); Mouse-Nestin (abclonal; Cat# - AB22035; IF concentration - 1:200); Rabbit-GFAP (Novus Biologicals; Cat# - NB300-141; IF concentration - 1:1000); Mouse-TubIII (Tuj1) (Millipore Sigma; Cat# - T8660: IF concentration - 1:500); Goat-DCX (Santa Cruz Biotechnology; Cat# - Sc-8066; IF concentration - 1:500).

Techniques: Expressing, Fluorescence, Staining, Labeling, Injection, Two Tailed Test, Control

Journal: Journal of neuropathology and experimental neurology

Article Title: Hereditary ferritinopathy: a novel mutation, its cellular pathology, and pathogenetic insights.

doi: 10.1093/jnen/64.4.280

Figure Lengend Snippet: FIGURE 9. Hyaline deposits. Pallidum. (A) Pleomorphic hyaline deposits, some containing nuclei, within the same area of GPe as in Figure 6A. (B) Prussian-blue reactivity. (C) PTAH reactivity. (D) Polyclonal anti-ferritin immunoreactivity. (E) Rare association of hyaline deposits with GFAP-immunoreactive astrocyte. (F) HO-2-immunoreactivity. Original magnifications: (A) Hematoxylin and eosin, 1503; (B) Perl’s stain, 2203; (C) 2203; (D) 2503; (E) 2203; (F) 2503.

Article Snippet: In order to identify the cell(s) of origin of the unique vacuolated nuclei, antibodies to S-100 (polyclonal, 1:20k, Lot #941; Innovation Foundation, Toronto, Ontario, Canada), GFAP, CD68 (monoclonal, 1:200, Lot #120201; DAKO), and carbonic anhydrase II (CAII) (polyclonal, 100-401-136/4383, 1:15k with retrieval; Rockland, Gilbertsville, PA) were applied to sections of basal ganglia and cerebellum, while antibodies to synaptophysin (monoclonal, 1:100 with retrieval, #M0363-UC/0403; Biogenex, San Ramon, CA), PGP 9.5 (polyclonal, 1:1,000 with retrieval, Lot #11830; Biogenesis, Handsown, NH), neurofilament protein 2F11 (monoclonal, 1:350, #M0762/117 DAKO) (101), and amyloid precursor protein (APP A4; monoclonal, 1:20K with heat retrieval, #23080547, Chemicon) were applied to the section of basal ganglia.

Techniques: Staining

Journal: Neural plasticity

Article Title: Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy.

doi: 10.1155/2015/676473

Figure Lengend Snippet: Figure 2: Repeated PTL administration influenced microglia/macrophage activation in the spinal cord level under neuropathic pain. Seven days after CCI in the ipsilateral dorsal spinal cord the protein level for IBA1 (a) and GFAP (b) were upregulated due to nerve injury. Repeated PTL administration increased the level of IBA1 (a), but the GFAP protein level was unchanged (b). The Western blot data are presented as the mean ± SEM and represent the normalized averages derived from analyses of 5–7 samples for each group. Intergroup differences were analyzed using ANOVA followed by Bonferroni’s multiple comparison test. ∗∗∗𝑃< 0.001 indicate significant differences compared to na¨ıve rats. #𝑃< 0.05 indicate significant differences between vehicle-treated and PTL-treated CCI-exposed rats. N-na¨ıve, V-vehicle, and PTL- parthenolide. The immunoblots shown are representative of 5–7 individual samples.

Article Snippet: Next, the blots were incubated with the following anti-rat primary antibodies that had been diluted in a SignalBoost Immunoreaction Enhancer Kit (Calbiochem) for 24 h at 4∘C: IBA1 (ProteinTech) 1 : 1000; GFAP (Novus Biologicals) 1 : 50000; IL-1β (Abcam) 1 : 1000; IL-6 (Invitrogen) 1 : 1000; iNOS (Sigma-Aldrich) 1 : 2000; IL-18 (R&D Systems) 1 : 1000; IL-10 (Invitrogen) 1 : 2000; TIMP1 (Novus Biologicals) 1 : 2000; pp38 MAPK (Cell Signalling) 1 : 2000; p-ERK1/2 (Santa Cruz) 1 : 4000; p-NF-κB (Santa Cruz) 1 : 2000; and p-STAT3 (Cell Signaling) 1 : 2000.

Techniques: Activation Assay, Western Blot, Derivative Assay, Comparison

Journal: Biomolecules & therapeutics

Article Title: PEGylated Erythropoietin Protects against Brain Injury in the MCAO-Induced Stroke Model by Blocking NF-κB Activation.

doi: 10.4062/biomolther.2019.147

Figure Lengend Snippet: Fig. 2. Effect of P-EPO on MDA and GSH/GSSG levels and expression of GFAP and Iba-1. Vehicle, 5000 U/kg of P-EPO, was adminis- tered via i.v. before MCAO. Ischemic brain tissues were processed for immunohistochemistry 24 h after reperfusion following MCAO. (A) MDA levels in the brain infarction site was measured using a TBARS assay kit. (B) GSH/GSSG levels in the brain infarction site were mea- sured using a GSH/GSSG ratio detection assay kit. Representative photomicrographs of (C) GFAP-positive astrocytes, (D) Iba-1-positive microglia/macrophages. Each value is presented as mean ± SD from five mice. **p<0.01 significant difference from MCAO mice. ##p<0.01, ###p<0.0001 significant difference from Sham mice (Sham n=6-7, MCAO n=8-9, M-EPO n=10-12).

Article Snippet: Membranes were incubated at room temperature for 2 h with the following specific antibodies: anti-COX-2, anti-IκB, anti-p-IκB, anti- STAT1, anti-p-STAT1, anti- STAT3, anti-pSTAT3, anti- STAT5, anti-p-STAT5 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-inducible nitric oxide synthase (iNOS) and anti-Glial fibrillary acidic protein (GFAP) (1:1,000 Novus Biologicals, Inc., Littleton, CO, USA), anti-p50, antip65, anti-BAX, anti-cleaved caspase-3, histone H1 (1:1,000; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), antihEPO (1:1,000, R&D systems, Inc., Minneapolis, MN), antiPEG-2-128, anti-PEG-B47 (1:1,000; Abcam, Cambridge, UK) and anti-β-actin (1:2,500; Sigma, St Louis, MO).

Techniques: Expressing, Immunohistochemistry, TBARS Assay, Detection Assay

Journal: Biomolecules & therapeutics

Article Title: PEGylated Erythropoietin Protects against Brain Injury in the MCAO-Induced Stroke Model by Blocking NF-κB Activation.

doi: 10.4062/biomolther.2019.147

Figure Lengend Snippet: Fig. 3. Effects of P-EPO on inflammatory protein and NF-κB, STAT5 activity. 10 h after reperfusion following 2-h MCAO, vehicle (Sham), 5,000 U/kg of P-EPO was administered via i.v. The ischemic cortex in the striatum was collected for Western blot or immunohistochemistry 24 h after reperfusion following MCAO. (A) Representative Western blots of iNOS, COX-2, GFAP and Iba-1 in the ischemic brain. (B) Rep- resentative Western blots of transcription factors STAT1, STAT3, and STAT5 and phosphorylated form and NF-κB factor p50 and p65. Equal loading was confirmed by monitoring the β-actin and histone-H1 protein levels. (C) The expression levels were quantified by stereological analysis using the ImageJ program. (D) Serum levels of cytokines after MCAO measured by ELISA. Anti-inflammatory cytokines (TNF-α, IL-6, IL-1β) were examined. The data are the mean ± SD each sample was probed in triplicate. n=5/group; *p<0.05, **p<0.01, ***p<0.0001 significant difference from MCAO mice. ##p<0.01, ###p<0.0001 significant difference from Sham mice (Sham n=8-9, MCAO n=8-9, M-EPO n=10-12).

Article Snippet: Membranes were incubated at room temperature for 2 h with the following specific antibodies: anti-COX-2, anti-IκB, anti-p-IκB, anti- STAT1, anti-p-STAT1, anti- STAT3, anti-pSTAT3, anti- STAT5, anti-p-STAT5 (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-inducible nitric oxide synthase (iNOS) and anti-Glial fibrillary acidic protein (GFAP) (1:1,000 Novus Biologicals, Inc., Littleton, CO, USA), anti-p50, antip65, anti-BAX, anti-cleaved caspase-3, histone H1 (1:1,000; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), antihEPO (1:1,000, R&D systems, Inc., Minneapolis, MN), antiPEG-2-128, anti-PEG-B47 (1:1,000; Abcam, Cambridge, UK) and anti-β-actin (1:2,500; Sigma, St Louis, MO).

Techniques: Activity Assay, Western Blot, Immunohistochemistry, Expressing, Enzyme-linked Immunosorbent Assay