rabbit anti-pitx3 polyclonal antibody  (Thermo Fisher)

Journal: Molecular Brain

Article Title: Histamine impairs midbrain dopaminergic development in vivo by activating histamine type 1 receptors

doi: 10.1186/s13041-014-0058-x

Figure Lengend Snippet: HA injection decreases dopaminergic neurons without affecting β-III Tubulin. (A) Coronal sections of the ventral midbrain from E14 vehicle- and HA-injected rat embryos stained to detect neurons (β-III Tubulin+) and dopaminergic phenotype (TH+); a representative section of a vehicle-injected embryo showing many TH-positive neurons while in a similar midbrain section from a representative HA-injected embryo less TH-positive cells are present. Nuclei were detected with Hoechst. Inner white squares in the merge images represent the higher magnifications shown at the bottom. Scale bars: 150 μm and 50 μm for the low-power and high-power pictures, respectively. (B) β-III Tubulin and TH expression by qRT-PCR from E14 vehicle- and HA-injected embryos, with β-III Tubulin presenting no change, and a significant decrease in TH caused by HA. **p < 0.01. (C) Densitometric analyses of the effect of HA administration on protein levels of β-III Tubulin and diverse factors involved in dopaminergic specification and phenotype. HA injection decreased the protein level of TH, Lmx1a, Lmx1b and Pitx3 in midbrain tissue from E14 HA-injected embryos without affecting the generation of neurons, compared to the vehicle-injected condition. Values were normalized to GAPDH signal. *p < 0.05.

Article Snippet: The following antibodies were used: rabbit anti-rat H 1 R polyclonal antibody (1:1500, Santa Cruz Biotechnology); goat anti-rat H 2 R polyclonal antibody (1:1500, Santa Cruz Biotechnology); rabbit anti-Lmx1a polyclonal antibody (1:100, Abcam); rabbit anti-Lmx1b polyclonal antibody (1:100, Abcam); rabbit polyclonal anti-tyrosine hydroxylase (TH, 1:1000; Pel-freez) and rabbit anti-Pitx3 polyclonal antibody (1:500, Zymed).

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

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: shRNA sequences of Pitx3

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: shRNA

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Vector map and sequencing results of the interference plasmid. (A) Vector GV102 map for constructing a plasmid carrying a sequence for Pitx3 interference. (B) Sequencing peak maps of sequences for Pitx3 interference. The upper, middle, and lower maps represent Sh-3568, Sh-3569, and Sh-3570 sequences, respectively.

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Plasmid Preparation, Sequencing

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Screening interference plasmids. (A) Cellular fluorescence 48 hours after transient transfection. Green cells indicate successful transfection of the plasmids. Scale bars: 50 μm. (B) Western blots for knockdown validation. The bars represent the ratio of optical density values of Pitx3 to β-actin. There was significant difference between the two groups ( P < 0.01, mean ± SD, n = 3, one-way analysis of variance and the least significant difference test). Control indicates the empty plasmid; Sh-3568, the Pitx3-Sh-3568 plasmid; Sh-3569, the Pitx3-Sh-3569 plasmid; and Sh-3570, the Pitx3-Sh-3570 plasmid, which were all transiently transfected into MES23.5 cells.

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Fluorescence, Transfection, Western Blot, Plasmid Preparation

Journal: Neural Regeneration Research

Article Title: Impact of Pitx3 gene knockdown on glial cell line-derived neurotrophic factor transcriptional activity in dopaminergic neurons

doi: 10.4103/1673-5374.213557

Figure Lengend Snippet: Effects of Pitx3 gene interference on glial cell line-derived neurotrophic factor (GDNF) transcriptional activity. GDNF-luciferase represents the cells transfected with the GDNF-luciferase plasmid alone. GDNF-luciferase + Pitx3-Sh-3570 represents the cells cotransfected with Pitx3-Sh-3570 and GDNF-luciferase plasmids. Transcriptional activity of GDNF decreased in the cells cotransfected with Pitx3-Sh-3570 and GDNF-luciferase plasmids; ** P < 0.01, vs . the GDNF-luciferase group (mean ± SD, n ≥ 3, independent sample t -test).

Article Snippet: After blocking, membranes were incubated with the primary antibodies anti-Pitx3 (rabbit polyclonal antibody; Sigma, St. Louis, MO, USA) and anti-β-actin (mouse monoclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then secondary antibodies (IRDye 680RD goat anti-rabbit and IRDye 800CW goat anti-mouse; LI-COR Biosciences, USA).

Techniques: Derivative Assay, Activity Assay, Luciferase, Transfection, Plasmid Preparation