primary antibody against green fluorescent protein (gfp)  (Abmart Inc)

Journal: Neural Regeneration Research

Article Title: Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures

doi: 10.4103/1673-5374.391302

Figure Lengend Snippet: Most Atpα -CMT2 flies have motor and sensory defects and an abnormal circadian neuron dendritic morphology. (A) Images of control, Atpα -CMT2, and mutant flies expressing nSybGFP in the neuromuscular junction of third instar larvae. Staining with anti-GFP antibody revealed the morphology of neuromuscular junctions. All Atpα -CMT2 and mutant flies showed a decrease in synapse number and length. We utilized an online post hoc power calculator (https://clincalc.com/stats/Power.aspx) to assess statistical power, with a false positive rate set at 0.05. Except for the statistical power of Atpα mut/+ in the synapse length group, which is 56.4%, the statistical power of the other significantly different groups exceed 83.5%. (B) Plots of synapse number (left) and length (right) in control, Atpα -CMT2, and mutant flies ( n = 6–10). One-way analysis of variance with Tukey’s multiple comparison test were used. (C) Images of control, Atpα -CMT2, and mutant brains with Class IV multidendritic sensory neurons in the third instar larval body wall visualized by ppk -GAL4-driven mCD8-GFP. Except for Atpα D580F/+ and Atpα mut/+ , all other heterozygous Atpα -CMT2 flies showed significantly reduced dendritic branching. (D) Plots of dendritic branch number in control, Atpα -CMT2, and mutant flies ( n = 15–20). Dendritic branch number was compared using the nonparametric Kruskal-Wallis test with Dunn’s multiple comparison test. The statistical powers of the significantly different groups are between 94.7% and 100%. (E) Left: Image of a circadian circuit in a control brain, visualized by staining with anti-PDF antibodies. sLNvs and lLNvs indicate small and large ventral lateral neurons. Right: The region analyzed. (F) Images of representative PDF dorsal axonal projections from control, Atpα -CMT2, and mutant flies taken during the early day (ZT2) and early night (ZT14). Apart from Atpα mut/+ , there was a significant decrease in axonal arbor complexity of the PDF circuit in Atpα -CMT2 flies. (G) Quantification of the total number of intersections between concentric rings and axonal projections at ZT2 and ZT14 ( n = 8–11). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparison test. The statistical powers of the significantly different groups are between 76.9% and 100%. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars represent SEM. Atpα : Na pump α subunit; CMT2: Charcot-Marie-Tooth disease type 2; Gal4: galactose-responsive transcription factor GAL4; GFP: green fluorescent protein; lLNv: large ventral lateral clock neurons; ns: not significant; PDF: pigment-dispersing factor; ppk: polyphosphate kinase; sLNv: small ventral lateral clock neurons; ZT: zeitgeber time.

Article Snippet: To visualize neuromuscular junctions in third instar larvae, D42 -Gal4,UAS-nSyb-GFP flies (Bloomington Drosophila Stock Center, Bloomington, IN, USA, Cat# 9263) were prepared and stained overnight at 4°C with a primary antibody against green fluorescent protein (GFP; 1:1000, Thermo Fisher Scientific, Waltham, MA, USA, Cat# A11120, RRID: AB_221568).

Techniques: Control, Mutagenesis, Expressing, Staining, Comparison

Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Representative images of livers from S100a8-Cre and Lrp8 f/f ; S100a8-Cre ( Lrp8 ΔNeu ) mice at endpoint, demonstrating increased tumor burden in Lrp8 ΔNeu mice. (b, c) Quantification of liver-to-body weight ratio (b) and tumor counts (c) in Lrp8 ΔNeu versus control groups, showing a significantly higher tumor burden in the Lrp8 ΔNeu group (n = 9 per group). (d) Flow cytometry analysis showing an increased percentage of neutrophils in the tumor microenvironment of Lrp8 ΔNeu mice compared to controls (n = 6 per group). (e, f) Flow cytometric analysis of senescence markers in tumor-infiltrating neutrophils from represent mice. Senescence-associated β-galactosidase (SA-β-Gal) positive neutrophils (e) and p21 + neutrophils (f) were significantly increased in Lrp8 ΔNeu mice compared to control group (n = 5-6 per group). (g, h) Flow cytometric analysis of neutrophils isolated from Lrp8 ΔNeu and control tumors. Representative histogram showing NIR-H 2 SE fluorescence, a hydrogen selenide probe, in neutrophils. Quantification of the percentage of NIR-H 2 SE + neutrophils (g) and mean fluorescence intensity (MFI) of NIR-H 2 SE in neutrophils (h), showing significantly reduced hydrogen selenide levels in neutrophils after Lrp8 knockout (n = 5–6 per group). (i) Quantitative RT-PCR analysis of senescence-associated markers Cdkn1a in tumor-infiltrating neutrophils (n = 5–6 per group). (j, k) mRNA expression levels of S100a8 and S100a9 significantly elevated in tumor-infiltrating neutrophils from Lrp8 ΔNeu mice (n = 5-6 per group). (l) Schematic representation of hydrodynamic tail vein injection (HDTVi) constructs. Mice were injected with constructs encoding oncogenic NRas G12V and myr-AKT along with a plasmid that encodes a green fluorescent protein (GFP) -Sepp1 fusion protein for tracking Sepp1 localization. (m) Representative immunofluorescence images of liver tissues showing GFP (green) indicating Sepp1 localization, S100A9 (red) indicating neutrophils, lymphocyte antigen 6 family member G (Ly6G; white) as another neutrophil marker, and 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Images demonstrate the colocalization of GFP with neutrophils, indicating that neutrophils uptake Sepp1. The lower row of images shows magnified views of the boxed regions, highlighting Sepp1 uptake by neutrophils. Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: Tumor sections were blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature, after which sections were incubated (overnight at 4 °C) in PBS-1% BSA containing a primary antibody against green fluorescent protein (GFP; 1:500, Abmart, Cat. M20004), Ly6G (1:200, Servicebio, Cat. GB11229), or S100A9 (1:500, R&D Systems, Cat. AF2065).

Techniques: Control, Flow Cytometry, Isolation, Fluorescence, Knock-Out, Quantitative RT-PCR, Expressing, Injection, Construct, Plasmid Preparation, Immunofluorescence, Marker, Two Tailed Test